Antibiotic resistance can evolve through sequential accumulation of multiple mutations1. To study such gradual evolution, we developed a selection device, the morbidostat, which continuously monitors bacterial growth and dynamically regulates drug concentrations such that the evolving population is constantly challenged. We analyzed evolutionary trajectories of Escherichia coli populations towards resistance to chloramphenicol, doxycycline, and trimethoprim. Over a period of ~20 days, resistance levels increased dramatically, with parallel populations showing similar phenotypic trajectories. Whole-genome sequencing revealed both drug-specific and drug-general genetic changes. Chloramphenicol and doxycycline resistance evolved through diverse combinations of mutations in genes involved in translation, transcription, and transport2. In contrast, trimethoprim resistance evolved in a stepwise manner1,3, through mutations restricted to the target enzyme dihydrofolate reductase (DHFR)4,5. Sequencing DHFR over time revealed that parallel populations not only evolved similar mutations, but also acquired them in similar order6. Uncovering such recurrent genotypic pathways may help the spread of resistance.

Revealing the genetic changes responsible for antibiotic resistance can be critical for developing novel antibiotic therapies. However, systematic studies correlating genotype to phenotype in the context of antibiotic resistance have been missing. In order to fill in this gap, we evolved 88 isogenic Escherichia coli populations against 22 antibiotics for 3 weeks. For every drug, two populations were evolved under strong selection and two populations were evolved under mild selection. By quantifying evolved populations’ resistances against all 22 drugs, we constructed two separate cross-resistance networks for strongly and mildly selected populations. Subsequently, we sequenced representative colonies isolated from evolved populations for revealing the genetic basis for novel phenotypes. Bacterial populations that evolved resistance against antibiotics under strong selection acquired high levels of cross-resistance against several antibiotics, whereas other bacterial populations evolved under milder selection acquired relatively weaker cross-resistance. In addition, we found that strongly selected strains against aminoglycosides became more susceptible to five other drug classes compared with their wild-type ancestor as a result of a point mutation on TrkH, an ion transporter protein. Our findings suggest that selection strength is an important parameter contributing to the complexity of antibiotic resistance problem and use of high doses of antibiotics to clear infections has the potential to promote increase of cross-resistance in clinics.

The centroid of a fluorophore can be determined within Ϸ1.5-nm accuracy from its focused image through fluorescence imaging with one-nanometer accuracy (FIONA). If, instead, the sample is moved away from the focus, the point-spread-function depends on both the position and 3D orientation of the fluorophore, which can be calculated by defocused orientation and position imaging (DOPI). DOPI does not always yield position accurately, but it is possible to switch back and forth between focused and defocused imaging, thereby getting the centroid and the orientation with precision. We have measured the 3D orientation and stepping behavior of single bifunctional rhodamine probes attached to one of the calmodulins of the light-chain domain (LCD) of myosin V as myosin V moves along actin. Concomitant with large and small steps, the LCD rotates and then dwells in the leading and trailing position, respectively. The probe angle relative to the barbed end of the actin (␤) averaged 128°while the LCD was in the leading state and 57°in the trailing state. The angular difference of 71°r epresents rotation of LCD around the bound motor domain and is consistent with a 37-nm forward step size of myosin V. When ␤ changes, the probe rotates ؎27°azimuthally around actin and then rotates back again on the next step. Our results remove degeneracy in angles and the appearance of nontilting lever arms that were reported.3D orientation ͉ lever arm ͉ single molecule ͉ fluorescence imaging with one-nanometer accuracy C omplementary conformational changes can be measured on single motor proteins by use of fluorescence imaging with one-nanometer accuracy (FIONA) (1-3) and by single-molecule fluorescence polarization microscopy (SMFP) (4). FIONA is a method in which the emission distribution of a single fluorophore is detected by using a charge-coupled device and fitted to a 2D Gaussian function to determine the position of the probe. The positional accuracy of the measurement, typically Ϸ1.5 nm, is generally limited by the number of collected photons (1-3). In contrast, SMFP is sensitive to the 3D orientation of a single dye's transition dipole moments (4). In SMFP, the dye is excited by multiple polarized beams, incident from different directions. The resulting emission is split with respect to its polarization and detected with avalanche photodiodes (APDs). However, by slightly defocusing the microscope objective and by using appropriate fitting routines, the defocused image of the probe can be used to determine both its position and its orientation (5-7). We call this technique defocused orientation and position imaging (DOPI). When the sample is deliberately moved Ϸ500 nm away from the best focus position, combinations of lobes and fringes appear on the charge-coupled device. These images are compared with calculated model images to obtain the best estimates of both 3D orientation and position of the probe. Because the image is spread out over a greater number of pixels in DOPI versus FIONA, DOPI inherently has poorer signal-tonoise ratio...