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ABSTRACT The fitness cost of an antibiotic resistance gene (ARG) can differ across host strains, creating refuges that allow the maintenance of an ARG in the absence of direct selection for its resistance phenotype. Despite the importance of such ARG-host interactions for predicting ARG dynamics, the basis of ARG fitness costs and their variability between hosts are not well understood. We determined the genetic basis of a host-dependent cost of a β-lactamase, blaTEM-116*, that conferred a significant cost in one Escherichia coli strain but was close to neutral in 11 other Escherichia spp. strains. Selection of a blaTEM-116*-encoding plasmid in the strain in which it initially had a high cost resulted in rapid and parallel compensation for that cost through mutations in a P1-like phage gene, relAP1. When the wild-type relAP1 gene was added to a strain in which it was not present and in which blaTEM-116* was neutral, it caused the ARG to become costly. Thus, relAP1 is both necessary and sufficient to explain blaTEM-116* costs in at least some host backgrounds. To our knowledge, these findings represent the first demonstrated case of the cost of an ARG being influenced by a genetic interaction with a phage gene. The interaction between a phage gene and a plasmid-borne ARG highlights the complexity of selective forces determining the maintenance and spread of ARGs and, by extension, encoding phage and plasmids in natural bacterial communities.IMPORTANCEAntibiotic resistance genes (ARGs) play a major role in the increasing problem of antibiotic resistance in clinically relevant bacteria. Selection of these genes occurs in the presence of antibiotics, but their eventual success also depends on the sometimes substantial costs they impose on host bacteria in antibiotic-free environments. We evolved an ARG that confers resistance to penicillin-type antibiotics in one host in which it did confer a cost and in one host in which it did not. We found that costs were rapidly and consistently reduced through parallel genetic changes in a gene encoded by a phage that was infecting the costly host. The unmutated version of this gene was sufficient to cause the ARG to confer a cost in a host in which it was originally neutral, demonstrating an antagonism between the two genetic elements and underlining the range and complexity of pressures determining ARG dynamics in natural populations.

ABSTRACT Transcription of bacterial genes is controlled by the coordinated action of cis- and trans-acting regulators. The activity and mode of action of these regulators can reflect different requirements for gene products in different environments. A well-studied example is the regulatory function that integrates the environmental availability of glucose and lactose to control the Escherichia coli lac operon. Most studies of lac operon regulation have focused on a few closely related strains. To determine the range of natural variation in lac regulatory function, we introduced a reporter construct into 23 diverse E. coli strains and measured expression with combinations of inducer concentrations. We found a wide range of regulatory functions. Several functions were similar to the one observed in a reference lab strain, whereas others depended weakly on the presence of cAMP. Some characteristics of the regulatory function were explained by the genetic relatedness of strains, indicating that differences varied on relatively short time scales. The regulatory characteristics explained by genetic relatedness were among those that best predicted the initial growth of strains following transition to a lactose environment, suggesting a role for selection. Finally, we transferred the lac operon, with the lacI regulatory gene, from five natural isolate strains into a reference lab strain. The regulatory function of these hybrid strains revealed the effect of local and global regulatory elements in controlling expression. Together, this work demonstrates that regulatory functions can be varied within a species and that there is variation within a species to best match a function to particular environments. IMPORTANCE The lac operon of Escherichia coli is a classic model for studying gene regulation. This study has uncovered features such as the environmental input logic controlling gene expression, as well as gene expression bistability and hysteresis. Most lac operon studies have focused on a few lab strains, and it is not known how generally those findings apply to the diversity of E. coli strains. We examined the environmental dependence of lac gene regulation in 20 natural isolates of E. coli and found a wide range of regulatory responses. By transferring lac genes from natural isolate strains into a common reference strain, we found that regulation depends on both the lac genes themselves and on the broader genetic background, indicating potential for still-greater regulatory diversity following horizontal gene transfer. Our results reveal that there is substantial natural variation in the regulation of the lac operon and indicate that this variation can be ecologically meaningful.

Large-scale genome rearrangements have been observed in cells adapting to various selective conditions during laboratory evolution experiments. However, it remains unclear whether these types of mutations can be stably maintained in populations and how they impact the evolutionary trajectories. Here we show that chromosomal rearrangements contribute to extremely high copper tolerance in a set of natural yeast strains isolated from Evolution Canyon (EC), Israel. The chromosomal rearrangements in EC strains result in segmental duplications in chromosomes 7 and 8, which increase the copy number of genes involved in copper regulation, including the crucial transcriptional activator CUP2 and the metallothionein CUP1. The copy number of CUP2 is correlated with the level of copper tolerance, indicating that increasing dosages of a single transcriptional activator by chromosomal rearrangements has a profound effect on a regulatory pathway. By gene expression analysis and functional assays, we identified three previously unknown downstream targets of CUP2: PHO84, SCM4, and CIN2, all of which contributed to copper tolerance in EC strains. Finally, we conducted an evolution experiment to examine how cells maintained these changes in a fluctuating environment. Interestingly, the rearranged chromosomes were reverted back to the wild-type configuration at a high frequency and the recovered chromosome became fixed in less selective conditions. Our results suggest that transposon-mediated chromosomal rearrangements can be highly dynamic and can serve as a reversible mechanism during early stages of adaptive evolution.

Determining pattern in the dynamics of population evolution is a long-standing focus of evolutionary biology. Complementing the study of natural populations, microbial laboratory evolution experiments have become an important tool for addressing these dynamics because they allow detailed and replicated analysis of evolution in response to controlled environmental and genetic conditions. Key findings include a tendency for smoothly declining rates of adaptation during selection in constant environments, at least in part a reflection of antagonism between accumulating beneficial mutations, and a large number of beneficial mutations available to replicate populations leading to significant, but relatively low genetic parallelism, even as phenotypic characteristics show high similarity. Together, there is a picture of adaptation as a process with a varied and largely unpredictable genetic basis leading to much more similar phenotypic outcomes. Increasing sophistication of sequencing and genetic tools will allow insight into mechanisms behind these and other patterns. [ABSTRACT FROM AUTHOR]

Background: Troglitazone (TGZ) is a potential anticancer agent. Little is known about the effect of this agent on cancer cell migration. Methods: Human ovarian carcinoma cell line, ES-2 cells were treated with various concentrations of TGZ. Cell migration was evaluated by wound-healing and Boyden chamber transwell experiments. PPARγ expression was blocked by PPARγ small interfering RNA. The effects of TGZ on phosphorylation of FAK, PTEN, Akt were assessed by immunoblotting using phospho-specific antibodies. The cellular distribution of paxillin, vinculin, stress fiber and PTEN was assessed by immunocytochemistry. Results: TGZ dose- and time-dependently impaired cell migration through a PPARγ independent manner. TGZ treatment impaired cell spreading, stress fiber formation, tyrosine phosphorylation of focal adhesion kinase (FAK), and focal adhesion assembly in cells grown on fibronectin substratum. TGZ also dose- and time-dependently suppressed FAK autophosphorylation and phosphorylation of the C-terminal of PTEN (a phosphatase). At concentration higher than 10 µM, TGZ caused accumulation of PTEN in plasma membrane, a sign of PTEN activation. Conclusion: These results indicate that TGZ can suppress cultured ES-2 cells migration. Our data suggest that the anti-migration potential of TGZ involves in regulations of FAK and PTEN activity. [ABSTRACT FROM AUTHOR]