Browsing by Subject "Horizontal gene transfer"

Lichens are symbioses between a fungus and a photosynthesizing partner such as a green alga or a cyanobacterium. Unlike mycorrhizal or rhizobial symbioses, the lichen symbiosis is not well understood either morphologically or molecularly. The lichen symbiosis has been somewhat neglected for several reasons. Lichens grow very slowly in nature (less than 1 cm a year), it is difficult to grow the fungus and the alga separately and, moreover, it remains difficult to resynthesize the mature symbiosis in the laboratory. It is not yet possible to delete genes, nor has any transformation method been established to introduce genes into the genomes of either the fungus or the alga. However, the lack of genetic tools for these organisms has been partially compensated for by the sequencing of the genomes of the lichenizing fungus Cladonia grayi and its green algal partner Asterochloris sp. This work uses the model lichen system Cladonia grayi and the associated genomes to explore one evolutionary and one developmental question concerning the lichen symbiosis.

Chapter One uses data from the genomes to assess whether there was evidence of horizontal gene transfer between the lichen symbionts in the evolution of this very intimate association; that is, whether genes of algal origin could be found in the fungal genome or vise versa. An initial homology search of the two genomes demonstrated that the fungus had, in addition to ammonium transporter/ammonia permease genes that were clearly fungal in origin, ammonium transporter/ammonia permease genes which appeared to be of plant origin. Using cultures of various lichenizing fungi, plant-like ammonium transporter/ammonia permease genes were identified by degenerate PCR in ten additional species of lichen in three classes of lichenizing fungi including the Lecanoromycetes, the Eurotiomycetes, and the Dothidiomycetes. Using the sequences of these transporter genes as well as data from publically available genome sequences of diverse organisms, I constructed a phylogy of 513 ammonium transporter/ammonia permease sequences from 191 genomes representing all main lineages of life to infer the evolutionary history of this family of proteins. In this phylogeny I detected several horizontal gene transfer events, including the aforementioned one which was demonstrated to be not a transfer from plants to fungi or vise versa, but a gene gain from a group of phylognetically unrelated hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina), which was subsequently lost in most lineages but retained in even distantly related lichenized fungi. Also demonstrated was the loss of the native fungal ammonium transporter and the subsequent replacement of this gene with a bacterial ammonium transporter during the early evolution of the fungi. Several additional recent horizontal gene transfers into lineages of eukaryotes were demonstrated as well. The phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). I show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: AMT family as defined here exhibits standard parent-to-offspring inheritance, whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs) into eukaryotes. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with ammonium transporters and ammonia permeases forming two distinct and possibly monophyletic groups.

In Chapter Two I address a follow-up question: in key lichenizing lineages for which ammonium transporter/ammonia permease (AMTP) genes were not found in Chapter One, were the genes lost? The only definitive infomation which can demonstrate absence of a gene from a genome is a full genome sequence. To this end, the genomes of eight additional lichenizing fungi in the key clades including the Caliciales (sensu Gaya 2011), the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales were sequenced using the Ilumina HiSeq technology and assembled with the short reads assembly software Velvet. These genomes were searched for ammonium transporter/ammonia permease sequences as well as 20 test genes to assess the completeness of each assembly. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacteria as a primary photobiont or living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the Acarosporomyetidae and Arthoniomycetes. In addition, the four AMTP genes from Cladonia grayi were shown to be functional by expression of the lichen genes in a strain of Saccharomyces cerevisiae in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source.

In Chapter Three I use genome data to address a developmental aspect of the lichen symbiosis. The finding that DNA in three genera of lichenizing fungi is methylated in symbiotic tissues and not methylated in aposymbiotic tissues or in the free-living fungus (Armaleo & Miao 1999a) suggested that epigenetic silencing may play a key role in the development of the symbiosis. Epigenetic silencing involves several steps that are conserved in many eukaryotes, including methylation of histone H3 at lysine 9 (H3K9) in nucleosomes within the silenced region, subsequent binding of heterochromatin-binding protein (HP1) over the region, and the recruitment of DNA methyltransferases to methylate the DNA, all of which causes the underlying chromatin to adopt a closed conformation, inhibiting the transcriptional machinery from binding. In this chapter I both identify the genes encoding the silencing machinery and determine the targets of the silencing machinery. I use degenerate PCR and genome sequencing to identify the genes encoding the H3K9 histone methyltransferase, the heterochromatin binding protein, and the DNA methyltransferases. I use whole genome bisulfite sequencing of DNA from the symbiotic structures of Cladonia grayi including podetia, squamules and soredia as well as DNA from cultures of the free-living fungus and free-living alga to determine which regions of the genome are methylated in the symbiotic and aposymbiotic states. In particular I examine regions of the genomes which appear to be differentially methylated in the symbiotic versus the aposymbiotic state. I show that DNA methylation is uncommon in the genome of the fungus in the symbiotic and aposymbiotic states, and that the genome of the alga is methylated in the symbiotic and aposymbiotic states.

Bioremediation is a treatment strategy that involves the removal of chemical pollutants using biological agents. When compared to physico-chemical treatment approaches, bioremediation causes less site disturbance and offers a range of other economic and ecological benefits. Yet, this treatment approach is not routinely selected mainly because of the unpredictability of the biological agents in the heterogeneous environments encountered at sites. Generally, three main approaches are utilized to improve bioremediation efficacy. These approaches consist of: 1) allowing native bacteria to degrade the pollutant (bioattenuation); 2) stimulating indigenous bacteria to improve their natural degradative capacity (biostimulation); or 3) supplying exogenous microorganisms that are known to degrade a specific contaminant if no known degraders are present at the site (bioaugmentation). Genetic bioaugmentation is another bioremediation treatment strategy, which relies on the well-studied mechanism of horizontal gene transfer (HGT) in which plasmids from exogenous donors are transferred to indigenous recipients. This strategy circumvents the need for the exogenous strain to compete with indigenous microorganisms under site conditions, a challenge that is difficult to overcome. HGT is a widespread natural phenomenon that readily occurs especially under harsh environmental conditions such as that in heavily contaminated environments.

Although all of these approaches show promise, in general, little is known about how organisms assemble in contaminated environments, limiting the implementation of bioremediation treatment strategies under field conditions. The work presented in this dissertation aims to address this challenge by characterizing and engineering prokaryotic microbiomes in soils contaminated with polycyclic aromatic hydrocarbons (PAHs). The overarching goals of this dissertation were to first utilize next-generation sequencing (NGS) to characterize pollutant-exposed environmental microbiomes and then use these data to develop a generalizable framework, which combines metagenomic and physico-chemical data to inform bioremediation treatment strategy selection. Then, in order to effectively validate this framework, a method for monitoring catabolic plasmid conjugation was developed. This dissertation was broadly broken down into four objectives as described below.

The first objective was to investigate environmentally relevant adaptation events in sediment microbial communities and gut-associated microbial communities in Atlantic killifish exposed to PAHs. Sediment and gut samples were obtained from the Republic Creosote Co. site located along the Elizabeth River (ER) and the Kings Creek (KC) reference site and their microbiomes were comparatively analyzed using high-throughput sequencing. In addition, because it is known gut microbes regulate host metabolism, the gut-associated metabolome was also characterized. Overall, significant community shifts were identified between sites, suggesting an environmental microbiome evolved to withstand high levels of PAHs. Specifically, of the OTUs identified, 9 species were different between KC guts and KC sediment while 176 were different between Republic guts and Republic sediment. These data suggest that factors other than dietary influence affect the microbiota colonized in the Republic fish gut. With respect to the metabolome, the amino acid (AA) concentrations were found to be higher for 19 out of 21 AAs in the Kings Creek samples when compared to the Republic samples. This indicates both a potential consequence of the microbial shifts and impact on metabolism between in the PAH-exposed fish sub-populations. Overall, this work provides insight into chemical-associated microbial community and metabolomics shifts and some of the potential resulting impacts.

The second objective was to develop a framework for precision bioremediation in which optimal microbial taxa could be identified for biostimulation, bioaugmentation and genetic bioaugmentation at a given site. Here, we developed an approach that combined Illumina Miseq high throughput sequencing, chemical profiling and Spearman correlation analyses. This framework was developed using samples obtained from the Holcomb Creosote Co. Superfund site, which is contaminated with both PAHs and heavy metals. Using this framework, Geobacter was identified as a biostimulation target while Mycobacterium and Sphingomonas were identified as strong potential biostimulation and genetic bioaugmentation targets, respectively. Based on availability and sequencing data, a consortium of Mycobacterium fredericksbergense, Sphingomonas aromaticivorans F199 (containing the pNL1 catabolic plasmid) was selected as a possible bioaugmentation cocktail to treat the Holcomb Creosote Superfund soils for PAHs. Though it is possible to identify prospective bioremediation targets using this methodology, this approach remains limited mainly due to the inadequate amount of fully sequenced site-specific environmental strains and catabolic plasmids available in databases. Additional research is needed especially using shotgun metagenomic sequencing, rather than amplicon-based sequencing, to increase the availability of environmental microbiome data and thereby improve the identification of potential donor strains and catabolic plasmids.

The third objective consisted of developing a method to effectively monitor genetic bioaugmentation and validate the method in a series of lab scale precision bioremediation scenarios. Previous methods used to monitor for HGT events have relied on using either fluorescent labeling or culturing. However, these monitoring techniques have been shown to be ineffective in complex matrices and for large-scale field applications. Herein, qPCR probes were designed to effectively monitor plasmid conjugation in complex matrices on a large scale.

In the final inclusive objective, the microbial cocktail formed by the framework developed in Objective 2 and the monitoring technique developed in Objective 3 were validated in lab scale reactors using a realistic PAH-contaminated soil matrix obtained from the Holcomb Creosote Co. Superfund site. Overall, it was found that the targeted microbial consortium was able to improve bioremediation by constructing an engineered environmental microbiome capable of increasing the rate of PAH biodegradation with a minimal long-term impact on the communities. In particular, there were overall increases in the class Bacilli and decreases in Betaproteobacteria sustained over time. In addition, the efforts to monitor genetic bioaugmentation were successful and HGT through plasmid conjugation was quantified. Specifically, the probes were able to detect conjugation of the NAH7 plasmid immediately following genetic bioaugmentation. Not surprisingly, the Inc-P9 plasmid was not maintained in the community, as it did not provide a strong enough consistent benefit towards survival to justify the metabolic load. The probes were also successful in quantifying the pNL1 plasmid, though no sustained HGT events were detected.

Overall, this dissertation provides significant advancements to the field of precision bioremediation. In particular, this dissertation work begins to integrate metagenomic and chemical measurements using statistical methods to effectively identify bioremediation targets and providing tools to monitor bioremediation progress under field relevant conditions. It is anticipated that as environmental microbiome databases continue to be populated, the use of frameworks such as that outlined in this dissertation work will be instrumental for the identification of targeted bioremediation treatment strategies.

Antibiotic resistance rates have increased in both clinical and environmental bacteria over the past several decades. While the causes underlying these trends have been investigated in a clinical setting, little work has been done to estimate the contribution of antibiotic resistance genes (ARGs) derived from non-traditional sources like transgenic (GM) crops. The cultivation and consumption of transgenic crops continues to be a widely debated topic, as the potential ecological impacts are not fully understood. In particular, because ARGs have historically been used as selectable markers in the genetic engineering of transgenic crops, it is important to determine if the genetic constructs found in decomposing or consumed transgenic crops persist long enough in the environment and if they can be transferred horizontally to indigenous microorganisms.

The first Objective of this dissertation addresses the question of persistence. Others have also estimated the DNA adsorption capacity of various clays, but have done so by manipulating the surface charge and size of particles tested which may overestimate sorption and underestimate the DNA available for horizontal transfer. In the present study, isotherms were generated using model Calf Thymus DNA and transgenic maize DNA without surface modification. Montmorillonite, kaolinite, and 3 soil mixtures with varying clay content were used in this study. The adsorption capacity of pure montmorillonite and kaolinite minerals was found to be one to two orders of magnitude less than previously estimated likely due to the distribution of clay particle sizes and heteroionic particle surface charge. However, it appears that a substantial amount of DNA is still able to adsorb onto these matrices (up to 200 mg DNA per gram of clay) suggesting the potential availability of free transgenic DNA in the environment may still be significant.

In addition to the soil environments described above, the persistence of these genes should be investigated in agricultural soils. Two important tools exist for understanding the potential contribution of transgenic crop-derived ARGs to environmental antibiotic resistance: (A) the microbiomes associated with transgenic maize and (B) decomposition behavior of transgenic crop biomass. The purpose of Objective Two is to characterize GM maize microbiomes and biomass decomposition to better how transgenic maize may be affecting antibiotic resistance rates among soil bacteria. To investigate this, bulk soil, rhizosphere soil, and internal endophyte prokaryotic microbiomes associated with conventional and transgenic maize were compared using lllumina MiSeq 16S amplicon sequencing. Internal endophytes were significantly influenced by time and location within the maize, but not by maize type. Nitrogen-cycling bacteria in the rhizosphere of transgenic pest-resistant (BT) maize experienced a decrease in diversity at day 56 of maize cultivation that was moderately correlated with the level of Cry1Ab protein exudates in soils surrounding transgenic BT maize. Secondly, bla ARG expression was tracked across 40 weeks of maize biomass decomposition in soils associated with conventional and transgenic BT maize. Bla expression significantly increased in soils associated with BT maize relative to conventional maize soils after 32 weeks or decomposition.

In addition to agricultural environments, wastewater treatment plants (WWTPs) may also contain genes derived from transgenic crops. Consumption of transgenic crops and foods containing them is common in the United States. Once ingested, DNA within transgenic crops can conceivably behave in three ways: 1) DNA will be degraded by acids or DNase I and II enzymes found within the digestive system, 2) DNA will be taken up by cells in the gut (host or bacterial), or 3) DNA will pass through the digestive system and excreted partially or wholly intact. In addition to degradation or uptake, transgenic DNA may be excreted as solid waste by the host. As free DNA contained within food matrices is protected from both enzymatic digestion and acid hydrolysis, the genes contained within foods containing GMOs may be allowed to pass through the host’s digestive tract intact and enter into WWTP environments. Objective 3 assessed this possibility by screening activated sludge and digester sludge obtained from France and the United States for the following four possible transgenes: p35 promoter gene, nos terminator gene, nptII ARG, and bla ARG. All wastewater samples were first screened using end-point PCR and all positive results were further analyzed with qRT-PCR to determine the relative concentration of each gene in wastewater. All domestic activated and digester sludge samples were found to contain at least one transgene, with the majority of samples containing all four. However, wastewater from France, where transgenic crops are neither cultivated nor consumed, contained significantly fewer copies of p35, nos, and nptII genes. Based on these findings, it is possible that some of these genes are derived from partially digested transgenic crop material present in human waste. This is supported in part by the significant differences in p35, nos, and nptII concentrations in French WWTP systems relative to domestic WWTP systems, as well as additional screening for Cauliflower Mosaic Virus (CaMV) ORF VII genes and larger fragments of possible transformation plasmids. Overall it is possible for genes contained within transgenic crops and processed foods containing their byproducts to survive the human digestive process and enter into WWTP environments, though the source of the detected genes could not be definitely confirmed.

The fate and behavior of these for genes in WWTPs remains largely uncharacterized. Further investigation of these trends in WWTP anaerobic digesters is of paramount importance, especially in the case of the bla and nptII ARGs, as a large fraction of WWTP-generated biosolids is used for land application. In addition, because of the relatively high background levels of antibiotic contaminants in wastewaters there is the further possibility that free ARGs may be horizontally transferred to WWTP bacteria, thus increasing antibiotic resistance in the environment. The ability of WWTP bacteria to take up these transgenes in this environment is a factor of (1) the observed rates of horizontal gene transfer (HGT) in anaerobic environments, (2) the abundance of genes of interest within WWTP anaerobic digesters, and (3) the presence of a selective pressure. Like most other topics related to HGT, observing and quantifying the rates of these event in anaerobic environments has proved to be a difficult task. To investigate the behavior of free ARG DNA in WWTP environments, anaerobic digesters were constructed using anaerobic feed sludge from the North Durham (NC) WWTP. Digesters belonged to one of the following treatments and were operated at 47oC in quadruplicate for a period of 60 days: 102 added bla and nptII transgene copies/mL, 104 added bla and nptII transgene copies/mL, or 106 added bla and nptII transgene copies/mL. Control digesters were also constructed. Across 60 days of operation, persistence of free DNA in anaerobic digester wastewater was significantly influenced by DNA fragment length. LUG genes significantly decreased after day 7, but nptII and bla ARGs persisted past 30 days of operation. Bla ARGs were only detected in iDNA within 106 transgene copies/mL treatments at day 60 of incubation. It is possible that this increase is due to bacterial uptake of free bla in eDNA as a result of an antibiotic-driven selective pressure, inducing the integration of bla into bacterial cells. NptII genes were below detection in iDNA at no time points after digester construction. Finally, the naturally occurring concentrations of bla and nptII ARGs in anaerobic digesters are consistent with the findings presented in Chapter 5. However, the existence of these ARGs as primarily eDNA is novel and presently unreported in the literature.

Antibiotic discovery and widespread usage has revolutionized the treatment of infectious diseases. However, this golden age of modern-day medicine is threatened by the increasing prevalence of antibiotic-resistant pathogens. As the antibiotic development pipeline increasingly slows, we find ourselves falling behind in the race between innovation and evolution.

Among the various means of bacterial evolution, horizontal gene transfer (HGT) – or the non-genealogical transmission of DNA between organisms – is the dominant mode responsible for the acquisition of antibiotic resistance genes. Combined with antibiotic overuse and misuse, HGT primarily via conjugation, has compromised the efficacy of nearly every single antimicrobial available. Tight coupling between HGT and antibiotic-mediated selection, along with a lack of quantitative experiments, has led to the general belief that antibiotics themselves promote gene transfer; however, antibiotic action could modulate the rate of gene transfer (as is assumed), the resulting population dynamics, or both. Therefore, it is critical to decouple these two processes to definitively determine the influence of antibiotics on conjugation.

In my dissertation, I quantified the extent to which antibiotics influence conjugation in the presence and absence of antibiotic-mediated selection dynamics. To do so, I implemented a synthetically engineered conjugation system, which facilitates precise quantification of conjugation dynamics. Using this platform, I quantified the rate of gene transfer, or the conjugation efficiency, in the absence of confounding selection dynamics. I discovered that, in contrast to conventional wisdom, antibiotics did not significantly increase the conjugation efficiency. This finding was general to 10 antibiotics, as well as nine native and clinically relevant plasmids. Instead, antibiotic selection dynamics alone could account for conjugation dynamics.

I next investigated the potential strategies to minimize, or ideally reverse, plasmid-mediated resistance. Traditionally, reducing overall antibiotic use has been the primary approach to reversing resistance; minimizing selection takes advantage of costly resistance genes to competitively displace resistant bacteria with their sensitive counterparts. However, despite widespread antibiotic stewardship initiatives, even costly resistance persists for long periods of time. One potential explanation is that sufficiently fast conjugation enables plasmid persistence in the absence of selection. Similar challenges in quantifying conjugation have prevented general conclusions, and overall, the extent to which conjugation enables persistence is unknown.

Using the same platform, I showed that conjugation enables the persistence of costly plasmids, even in the absence of selection. Conjugation-assisted persistence was true for nine common conjugal plasmids, and in microbial populations consisting of varying degrees of complexity. Finally, I showed that by reducing the conjugation efficiency and promoting plasmid loss, it is possible to reverse resistance. Together, these findings contribute to basic understanding of the propagation and persistence plasmids, and elucidate a novel therapeutic strategy by taking advantage of ecological/evolutionary dynamics to to reduce or even reverse the spread of resistance.

Plants use an array of photoreceptors to measure the quality, quantity, and direction of light in order to respond to ever-changing light environments. Photoreceptors not only determine how and when individual plants complete their life cycles, but they also have a profound and long-term macroevolutionary influence on species diversification. Despite their significances, very little is known about photoreceptors across plants as whole, and we lack a comprehensive view of photoreceptor evolution.

In my dissertation, I investigate the origin and evolution of three of the most prominent photoreceptor gene families in plants: phytochromes, phototropins and neochromes. Using newly available transcriptomic and genomic data, I completed the first in-depth survey of these photoreceptor families across land plants, green algae, red algae, glaucophytes, cryptophytes, haptophytes, and stramenopiles.

Phytochromes are red/far-red photoreceptors that play essential roles in seed germination, seedling photomorphogenesis, shade-avoidance, dormancy, circadian rhythm, phototropism, and flowering. Here, I show that the canonical plant phytochromes originated in a common ancestor of streptophytes (charophyte green algae plus land plants), and I identify the most likely sequence whereby the plant phytochrome structure evolved from its ancestral phytochrome. Phytochromes in charophyte algae are structurally diverse, including canonical and non-canonical forms, whereas in land plants, phytochrome structure is highly conserved. Liverworts, hornworts, and Selaginella apparently possess a single phytochrome gene copy, whereas independent gene duplications occurred within mosses, lycopods, ferns, and seed plants, leading to diverse phytochrome families in these clades. My detailed phylogeny encompasses all of green plants and enables me to not only uncover new phytochrome lineages, but also to make links to our current understanding of phytochrome function in Arabidopsis and Physcomitrella (the major model organism outside of flowering plants). Based on this robust evolutionary framework, I propose new hypotheses and discuss future directions to study phytochrome mechanisms.

Phototropins are blue-light photoreceptors that regulate key adaptive physiological responses, including shoot-positive phototropism, root-negative phototropism, chloroplast accumulation/avoidance, stomatal opening, circadian rhythm, leaf expansion, and seedling elongation I show that phototropins originated in the common ancestor of Viridiplantae (all green algae [charophytes, chlorophytes, prasinophytes] plus land plants). Phototropins repeatedly underwent independent duplications in all major plant lineages (mosses, lycopods, ferns and seed plants), except for liverworts and hornworts, where phototropin is a single-copy gene. Following each major duplication event, phototropins subsequently differentiated in parallel, resulting in two specialized (yet partially overlapping) functional forms that primarily mediate either low- or high-light responses. My gene phylogeny further suggests that phototropins have co-evolved with phytochromes, as is evident from their molecular interactions and strikingly similar gene duplication patterns. I hypothesize that the co-evolution of phototropins with phytochromes, together with their subsequent convergent functional divergences in phototropic responses, contributed to the success of plants in adapting to diverse and heterogeneous habitats.

Neochromes are chimeric photoreceptors that, by fusing phytochrome and phototropin modules into a single protein, are able to use both red/far-red and blue light to modulate phototropic responses. Neochromes were first discovered in ferns, and the evolution of neochromes was implicated as a key innovation that facilitated fern diversification under the low-light angiosperm canopies. Despite its significance from an evolutionary standpoint, the origin of neochromes has remained a mystery. Here I present the first evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in my large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 MYA, long after the split between the two plant lineages (at least 400 MYA). By analyzing the draft genome of the Anthoceros punctatus hornwort, I also discovered a novel phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was horizontally transferred to ferns, where it may have played a significant role in the diversification of modern ferns.

In summary, my studies identified the molecular origins of phytochromes, phototropins and neochromes, and reconstructed their respective evolutionary histories. This new framework for photoreceptor evolution will stimulate new research linking ecology, evolution, and photochemistry to understand how plants adapt to variable light environments.

Plasmids are key vehicles of horizontal gene transfer (HGT), mobilizing antibiotic resistance, virulence, and other traits among bacterial populations. Their fitness is directed by two orthogonal processes—vertical transfer through cell division and horizontal transfer through conjugation. When considered individually, improvements in either mode of transfer can promote how well a plasmid spreads and persists. Together, however, the metabolic burden of conjugation could create a tradeoff between the two and constrain plasmid evolution. Furthermore, the environmental and genetic forces that drive plasmid transfer are poorly understood, due to the lack of definitive quantification coupled with genomic analysis. Knowing how conjugation influences plasmid fitness and, subsequently, how to influence conjugation opens the door for evolutionary therapies targeting antibiotic resistance and bacteria in general.

Here, we integrate conjugative phenotype with plasmid genotype to provide quantitative analysis of HGT in clinical Escherichia coli pathogens. We find a substantial proportion of these pathogens (>25%) able to readily spread resistance to the most common classes of antibiotics. Antibiotics of varied modes of action had less than a 5-fold effect on conjugation efficiency in general, with one exception displaying 31-fold promotion upon exposure to macrolides and chloramphenicol. In contrast, genome sequencing reveals plasmid incompatibility group strongly correlates with transfer efficiency. These findings offer new insights into the determinants of plasmid mobility and have implications for the development of treatments that target HGT.

We also present evidence for the presence, consequences, and molecular basis of a conjugation-burden tradeoff among 40 plasmids derived from clinical E. coli pathogens. We discover that most plasmids avoid a fitness tradeoff by operating below a conjugation efficiency threshold for burden, indicating strong selection for vertical transfer. In this region, E. coli demonstrates a remarkable growth tolerance to over four orders of magnitude change in conjugation efficiency. This tolerance fades as nutrients become scarce and horizontal transfer attracts a larger share of host resources. These results provide insight into evolutionary constraints directing plasmid fitness and strategies to combat the spread of antibiotic resistance.

Conjugative plasmids can mediate the spread and maintenance of diverse traits and functions in microbial communities. This role depends on the plasmid’s ability to persist in a population. However, for a community consisting of multiple populations transferring multiple plasmids, the conditions underlying plasmid persistence are poorly understood. Here, I described a plasmid-centric framework that makes it computationally feasible to analyze gene flow in complex communities. Using this framework, I derived the ‘persistence potential’: a general, heuristic metric that predicts the persistence and abundance of any plasmids. I validated the metric with engineered microbial consortia transferring mobilizable plasmids and with quantitative data available in the literature. I used the learned principles governing plasmid persistence to propose a new strategy to control community functional stability. The functions of many microbial communities exhibit remarkable stability despite fluctuations in the compositions of these communities. To date, a mechanistic understanding of this function-composition decoupling is lacking. Statistical mechanisms have been commonly hypothesized to explain such decoupling. Here, I proposed that dynamic mechanisms, mediated by horizontal gene transfer (HGT), also enable the independence of functions from the compositions of microbial communities. I combined theoretical analysis with numerical simulations to illustrate that HGT rates can determine the decoupling and functional stability of microbial communities. I and my collaborator further validated these predictions using engineered microbial consortia of different complexities transferring one or more than a dozen clinically isolated plasmids, as well as through the re-analysis of data from the literature. Next, I applied the general principles underlying plasmid persistence to understand how population bottlenecks affect plasmid fates. Natural microbiomes often experience population bottlenecks, which create heterogenous local communities, each with reduced population size and biodiversity. Based on the concept of ‘persistence potential’, I and my collaborators demonstrated that bottlenecks can paradoxically promote the persistence of a plasmid that would otherwise not persist in a well-mixed global community. In particular, among the local communities created by a bottleneck, a minority will primarily consist of members able to transfer the plasmid fast enough to support its maintenance. They serve as a local haven that protects the plasmid from being eliminated. Our results provide new insights into the microbiome functional stability and suggest a generalizable approach to modulate plasmid maintenance in complex communities. These works will facilitate a quantitative understanding of natural microbial communities and the engineering of microbial consortia.