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Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility. Cytosine modifications are important epigenetic markers yet their physical influence on DNA is not well understood. Here, Ngo et al . show that different alterations affect DNA flexibility, suggesting a mechanism where modifications change accessibility of nucleosome bound DNA.

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli . Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer. Significance Understanding how bacteria rapidly evolve under antibiotic selective pressure is crucial to controlling the development of resistant organisms. We show that initial resistance emerges from successful segregation of mutant chromosomes at the tips of filaments followed by budding of resistant progeny. We propose that the first stages of emergence of resistance occur via the generation of multiple chromosomes within the filament and are achieved by mutation and possibly recombination between the chromosomes.

The emergence of bacterial antibiotic resistance is a growing problem, yet the variables that influence the rate of emergence of resistance are not well understood. In a microfluidic device designed to mimic naturally occurring bacterial niches, resistance of Escherichia coli to the antibiotic ciprofloxacin developed within 10 hours. Resistance emerged with as few as 100 bacteria in the initial inoculation. Whole-genome sequencing of the resistant organisms revealed that four functional single-nucleotide polymorphisms attained fixation. Knowledge about the rapid emergence of antibiotic resistance in the heterogeneous conditions within the mammalian body may be helpful in understanding the emergence of drug resistance during cancer chemotherapy.

One of the physiological responses of bacteria to external stress is to assemble into a biofilm. The formation of a biofilm greatly increases a bacterial population's resistance to a hostile environment by shielding cells, for example, from antibiotics. In this paper, we describe the conditions necessary for the emergence of biofilms in natural environments and relate them to the emergence of biofilm formation inside microfluidic devices. We show that competing species of Escherichia coli bacteria form biofilms to spatially segregate themselves in response to starvation stress, and use in situ methods to characterize the physical properties of the biofilms. Finally, we develop a microfluidic platform to study the inter-species interactions and show how biofilm-mediated genetic interactions can improve a species' resistance to external stress.

We use a microfabricated ecology with a doxorubicin gradient and population fragmentation to produce a strong Darwinian selective pressure that drives forward the rapid emergence of doxorubicin resistance in multiple myeloma (MM) cancer cells. RNA sequencing of the resistant cells was used to examine (i) emergence of genes with high de novo substitution densities (i.e., hot genes) and (ii) genes never substituted (i.e., cold genes). The set of cold genes, which were 21% of the genes sequenced, were further winnowed down by examining excess expression levels. Both the most highly substituted genes and the most highly expressed never-substituted genes were biased in age toward the most ancient of genes. This would support the model that cancer represents a revision back to ancient forms of life adapted to high fitness under extreme stress, and suggests that these ancient genes may be targets for cancer therapy.

We have fabricated nanocomposites consisting of 4-Å carbon nanotubes embedded in the 0.7-nm pores of aluminophosphate-five (AFI) zeolite that display a superconducting specific heat transition at 15 K. MicroRaman spectra of the samples show strong and spatially uniform radial breathing mode (RBM) signals at 510 cm⁻¹ and 550 cm⁻¹, characteristic of the (4, 2) and (5, 0) nanotubes, respectively. The specific heat transition is suppressed at >2 T, with a temperature dependence characteristic of finite-size effects. Comparison with theory shows the behavior to be consistent with that of a type II BCS superconductor, characterized by a coherence length of 14 ± 2 nm and a magnetic penetration length of 1.5 ± 0.7 μm. Four probe and differential resistance measurements have also indicated a superconducting transition initiating at 15 K, but the magnetoresistance data indicate the superconducting network to be inhomogeneous, with a component being susceptible to magnetic fields below 3 T and other parts capable of withstanding a magnetic field of 5 T or beyond.

We present electrical and thermal specific heat measurements that show superconductivity in double-wall carbon nanotube (DWCNT) bundles. Clear evidence, comprising a resistance drop as a function of temperature, magnetoresistance and differential resistance signature of the supercurrent, suggest an intrinsic superconducting transition below 6.8 K for one particular sample. Additional electrical data not only confirm the existence of superconductivity, but also indicate the T c distribution that can arise from the diversity in the diameter and chirality of the DWCNTs. A broad superconducting anomaly is observed in the specific heat of a bulk DWCNT sample, which yields a T c distribution that correlates well with the range of the distribution obtained from the electrical data. As quasi one dimensionality of the DWCNTs dictates the existence of electronic density of state peaks, confirmation of superconductivity in this material system opens the exciting possibility of tuning the T c through the application of a gate voltage.

Base-pairing interactions between nucleic acids mediate target recognition in many biological processes. We developed a super-resolution imaging and modeling platform that enabled the in vivo determination of base pairing–mediated target recognition kinetics. We examined a stress-induced bacterial small RNA, SgrS, which induces the degradation of target messenger RNAs (mRNAs). SgrS binds to a primary target mRNA in a reversible and dynamic fashion, and formation of SgrS-mRNA complexes is rate-limiting, dictating the overall regulation efficiency in vivo. Examination of a secondary target indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other small RNA systems and other target search processes.

Base-pairing interactions between nucleic acids mediate target recognition in many biological processes. We developed a super-resolution imaging and modeling platform that enabled the in vivo determination of base pairing-mediated target recognition kinetics. We examined a stress-induced bacterial small RNA, SgrS, which induces the degradation of target mRNAs. SgrS binds to a primary target mRNA in a reversible and dynamic fashion, and formation of SgrS-mRNA complexes is rate-limiting, dictating the overall regulation efficiency in vivo. Examination of a secondary target indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other sRNA systems and other target search processes.

The emergence of bacterial antibiotic resistance is a growing problem. Many antibiotic drugs fail because resistant mutants evolve and populate again, as seen by the global spread of the infectious pathogens that cause diseases like tuberculosis. In the course of evolution in human body, bacteria encounter heterogeneous environment full of transient chemical and nutrient gradients. Antibiotic gradients can arise when a patient begins and ends therapies, or forgets doses. Bacteria have successfully adapted to a wide variety of physical/chemical conditions, suggesting that spatial heterogeneity must play an important role in evolution. However, the space dimension was under-appreciated by previous laboratory models of microbial evolution. Such observations motivate us to design a novel reactor that mimics naturally occurring heterogeneous ecology. Chapter 2 shows the reactor “Death Galaxy” that we make by microfabrication and microfluidic techniques. We find that the resistance of model bacterial Escherichia coli to the broad-spectrum antibiotic ciprofloxacin develops within 10 hours. Resistance emerges with as few as 100 bacteria in the initial inoculation. Whole-genome sequencing of the resistant organisms reveals that four functional single-nucleotide polymorphisms attain fixation. We further investigate how each factor involved in “Death Galaxy” - antibiotic gradient, motility, metapopulations - affects the rate of evolution, with both numerical simulations and experiments. The major role of heterogeneous environment is to increase the fixation rate of the resistant mutant. But how can the mutant emerge quickly when a sensitive population encounters antibiotics? In chapter 3, we zoom in to the single cell level and search for the origin of mutants. We find that the Escherichia coli is able to develop single cell mesoscopic ecological niches as a response to external antibiotics, where chromosomal replication can proceed to generate mutants. We then show that such strategy is implemented by the present order of genes placed on the chromosome with respect to the origin of replication. Finally, in chapter 4, we explore how interactions between individual genome affect the rate of evolution. We propose an evolutionary game theory model to calculate the conditions for horizontal gene transfer to be stable strategy. We further suggest horizontal gene transfer could play crucial role in microbial speciation through analyzing the genomic structure of closed bacterial species.