Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost.

Plasmids play a major role in rapid adaptation of bacteria by facilitating horizontal transfer of diverse genes, most notably those conferring antibiotic resistance. While most plasmids that replicate in a broad range of bacteria also persist well in diverse hosts, there are exceptions that are poorly understood. We investigated why a broad-host range plasmid, pBP136, originally found in clinical Bordetella pertussis isolates, quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution we found that inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid maintenance in E. coli. This gene inactivation resulted in decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. It also caused transcriptional changes in many chromosomal genes primarily related to metabolism. In silico analyses suggested that the change in plasmid transcriptome may be initiated by Upf31 interacting with the plasmid regulator KorB. Expression of upf31 in trans negatively affected persistence of pBP136Δupf31 as well as the closely related archetypal IncP-1ß plasmid R751, which is stable in E. coli and natively encodes a truncated upf31 allele. Our results demonstrate that while the upf31 allele in pBP136 might advantageously modulate gene expression in its original host, B. pertussis, it has harmful effects in E. coli. Thus, evolution of a single plasmid gene can change the range of hosts in which that plasmid persists, due to effects on the regulation of plasmid gene transcription.

Evolutionary "Crowdsourcing": Alignment of Fitness Landscapes Allows for Cross-species Adaptation of a Horizontally Transferred Gene.

Genes that undergo horizontal gene transfer (HGT) evolve in different genomic backgrounds. Despite the ubiquity of cross-species HGT, the effects of switching hosts on gene evolution remains understudied. Here, we present a framework to examine the evolutionary consequences of host-switching and apply this framework to an antibiotic resistance gene commonly found on conjugative plasmids. Specifically, we determined the adaptive landscape of this gene for a small set of mutationally connected genotypes in 3 enteric species. We uncovered that the landscape topographies were largely aligned with minimal host-dependent mutational effects. By simulating gene evolution over the experimentally gauged landscapes, we found that the adaptive evolution of the mobile gene in one species translated to adaptation in another. By simulating gene evolution over artificial landscapes, we found that sufficient alignment between landscapes ensures such "adaptive equivalency" across species. Thus, given adequate landscape alignment within a bacterial community, vehicles of HGT such as plasmids may enable a distributed form of genetic evolution across community members, where species can "crowdsource" adaptation.

Guidelines for the estimation and reporting of plasmid conjugation rates.

Conjugation is a central characteristic of plasmid biology and an important mechanism of horizontal gene transfer in bacteria. However, there is little consensus on how to accurately estimate and report plasmid conjugation rates, in part due to the wide range of available methods. Given the similarity between approaches, we propose general reporting guidelines for plasmid conjugation experiments. These constitute best practices based on recent literature about plasmid conjugation and methods to measure conjugation rates. In addition to the general guidelines, we discuss common theoretical assumptions underlying existing methods to estimate conjugation rates and provide recommendations on how to avoid violating these assumptions. We hope this will aid the implementation and evaluation of conjugation rate measurements, and initiate a broader discussion regarding the practice of quantifying plasmid conjugation rates.

Estimating the transfer rates of bacterial plasmids with an adapted Luria-Delbrück fluctuation analysis.

To increase our basic understanding of the ecology and evolution of conjugative plasmids, we need reliable estimates of their rate of transfer between bacterial cells. Current assays to measure transfer rate are based on deterministic modeling frameworks. However, some cell numbers in these assays can be very small, making estimates that rely on these numbers prone to noise. Here, we take a different approach to estimate plasmid transfer rate, which explicitly embraces this noise. Inspired by the classic fluctuation analysis of Luria and Delbrück, our method is grounded in a stochastic modeling framework. In addition to capturing the random nature of plasmid conjugation, our new methodology, the Luria-Delbrück method ("LDM"), can be used on a diverse set of bacterial systems, including cases for which current approaches are inaccurate. A notable example involves plasmid transfer between different strains or species where the rate that one type of cell donates the plasmid is not equal to the rate at which the other cell type donates. Asymmetry in these rates has the potential to bias or constrain current transfer estimates, thereby limiting our capabilities for estimating transfer in microbial communities. In contrast, the LDM overcomes obstacles of traditional methods by avoiding restrictive assumptions about growth and transfer rates for each population within the assay. Using stochastic simulations and experiments, we show that the LDM has high accuracy and precision for estimation of transfer rates compared to the most widely used methods, which can produce estimates that differ from the LDM estimate by orders of magnitude.

Coevolution of host-plasmid pairs facilitates the emergence of novel multidrug resistance.

Multidrug resistance (MDR) of pathogens is an ongoing public health crisis exacerbated by the horizontal transfer of antibiotic resistance genes via conjugative plasmids. Factors that stabilize these plasmids in bacterial communities contribute to an even higher incidence of MDR, given the increased likelihood that a host will already contain a plasmid when it acquires another through conjugation. Here, we show one such stabilizing factor is host-plasmid coevolution under antibiotic selection, which facilitated the emergence of MDR via two distinct plasmids in communities consisting of Escherichia coli and Klebsiella pneumoniae once antibiotics were removed. In our system, evolution promoted greater stability of a plasmid in its coevolved host. Further, pleiotropic effects resulted in greater plasmid persistence in both novel host-plasmid combinations and, in some cases, multi-plasmid hosts. This evolved stability favoured the generation of MDR cells and thwarted their loss within communities with multiple plasmids. By selecting for plasmid persistence, the application of antibiotics may promote MDR well after their original period of use.

Trait mapping in diverse arthropods by bulked segregant analysis.

Bulked segregant analysis (BSA) is a cross-based method for genetic mapping in sexually reproducing organisms. The method's use of bulked (pooled) samples markedly reduces the genotyping effort associated with traditional linkage mapping studies. Further, it can be applied to species with life histories or physical attributes (as for micro-insects) that render genetic mapping with other methods impractical. Recent studies in both insects and mites have revealed that advanced BSA experimental designs can resolve causal loci to narrow genomic intervals, facilitating follow-up investigations. As high-quality genomes become more widely available, BSA methods are poised to become an increasingly important tool for the rapid mapping of both monogenic and polygenic traits in diverse arthropod species.

Convergent evolution of cytochrome P450s underlies independent origins of keto-carotenoid pigmentation in animals.

Keto-carotenoids contribute to many important traits in animals, including vision and coloration. In a great number of animal species, keto-carotenoids are endogenously produced from carotenoids by carotenoid ketolases. Despite the ubiquity and functional importance of keto-carotenoids in animals, the underlying genetic architectures of their production have remained enigmatic. The body and eye colorations of spider mites (Arthropoda: Chelicerata) are determined by ß-carotene and keto-carotenoid derivatives. Here, we focus on a carotenoid pigment mutant of the spider mite Tetranychus kanzawai that, as shown by chromatography, lost the ability to produce keto-carotenoids. We employed bulked segregant analysis and linked the causal locus to a single narrow genomic interval. The causal mutation was fine-mapped to a minimal candidate region that held only one complete gene, the cytochrome P450 monooxygenase CYP384A1, of the CYP3 clan. Using a number of genomic approaches, we revealed that an inactivating deletion in the fourth exon of CYP384A1 caused the aberrant pigmentation. Phylogenetic analysis indicated that CYP384A1 is orthologous across mite species of the ancient Trombidiformes order where carotenoids typify eye and body coloration, suggesting a deeply conserved function of CYP384A1 as a carotenoid ketolase. Previously, CYP2J19, a cytochrome P450 of the CYP2 clan, has been identified as a carotenoid ketolase in birds and turtles. Our study shows that selection for endogenous production of keto-carotenoids led to convergent evolution, whereby cytochrome P450s were independently co-opted in vertebrate and invertebrate animal lineages.

High-resolution QTL mapping in Tetranychus urticae reveals acaricide-specific responses and common target-site resistance after selection by different METI-I acaricides.

Arthropod herbivores cause dramatic crop losses, and frequent pesticide use has led to widespread resistance in numerous species. One such species, the two-spotted spider mite, Tetranychus urticae, is an extreme generalist herbivore and a major worldwide crop pest with a history of rapidly developing resistance to acaricides. Mitochondrial Electron Transport Inhibitors of complex I (METI-Is) have been used extensively in the last 25 years to control T. urticae around the globe, and widespread resistance to each has been documented. METI-I resistance mechanisms in T. urticae are likely complex, as increased metabolism by cytochrome P450 monooxygenases as well as a target-site mutation have been linked with resistance. To identify loci underlying resistance to the METI-I acaricides fenpyroximate, pyridaben and tebufenpyrad without prior hypotheses, we crossed a highly METI-I-resistant strain of T. urticae to a susceptible one, propagated many replicated populations over multiple generations with and without selection by each compound, and performed bulked segregant analysis genetic mapping. Our results showed that while the known H92R target-site mutation was associated with resistance to each compound, a genomic region that included cytochrome P450-reductase (CPR) was associated with resistance to pyridaben and tebufenpyrad. Within CPR, a single nonsynonymous variant distinguished the resistant strain from the sensitive one. Furthermore, a genomic region linked with tebufenpyrad resistance harbored a non-canonical member of the nuclear hormone receptor 96 (NHR96) gene family. This NHR96 gene does not encode a DNA-binding domain (DBD), an uncommon feature in arthropods, and belongs to an expanded family of 47 NHR96 proteins lacking DBDs in T. urticae. Our findings suggest that although cross-resistance to METI-Is involves known detoxification pathways, structural differences in METI-I acaricides have also resulted in resistance mechanisms that are compound-specific.

Long-Term Population Studies Uncover the Genome Structure and Genetic Basis of Xenobiotic and Host Plant Adaptation in the Herbivore Tetranychus urticae.

Pesticide resistance arises rapidly in arthropod herbivores, as can host plant adaptation, and both are significant problems in agriculture. These traits have been challenging to study as both are often polygenic and many arthropods are genetically intractable. Here, we examined the genetic architecture of pesticide resistance and host plant adaptation in the two-spotted spider mite, Tetranychus urticae, a global agricultural pest. We show that the short generation time and high fecundity of T. urticae can be readily exploited in experimental evolution designs for high-resolution mapping of quantitative traits. As revealed by selection with spirodiclofen, an acetyl-CoA carboxylase inhibitor, in populations from a cross between a spirodiclofen-resistant and a spirodiclofen-susceptible strain, and which also differed in performance on tomato, we found that a limited number of loci could explain quantitative resistance to this compound. These were resolved to narrow genomic intervals, suggesting specific candidate genes, including acetyl-CoA carboxylase itself, clustered and copy variable cytochrome P450 genes, and NADPH cytochrome P450 reductase, which encodes a redox partner for cytochrome P450s. For performance on tomato, candidate genomic regions for response to selection were distinct from those responding to the synthetic compound and were consistent with a more polygenic architecture. In accomplishing this work, we exploited the continuous nature of allele frequency changes across experimental populations to resolve the existing fragmented T. urticae draft genome to pseudochromosomes. This improved assembly was indispensable for our analyses, as it will be for future research with this model herbivore that is exceptionally amenable to genetic studies.