Biogenic Control of Manganese Doping in Zinc Sulfide Nanomaterial Using Shewanella oneidensis MR-1

Bacteria naturally alter the redox state of many compounds and perform atom-by-atom nanomaterial synthesis to create many inorganic materials. Recent advancements in synthetic biology have spurred interest in using biological systems to manufacture nanomaterials, implementing biological strategies to specify the nanomaterial characteristics such as size, shape, and optical properties. Here, we combine the natural synthetic capabilities of microbes with engineered genetic control circuits toward biogenically synthesized semiconductor nanomaterials. Using an engineered strain of Shewanella oneindensis with inducible expression of the cytochrome complex MtrCAB, we control the reduction of manganese (IV) oxide. Cytochrome expression levels were regulated using an inducer molecule, which enabled precise modulation of dopant incorporation into manganese doped zinc sulfide nanoparticles (Mn:ZnS). Thereby, a synthetic gene circuit controlled the optical properties of biogenic quantum dots. These biogenically assembled nanomaterials have similar physical and optoelectronic properties to chemically synthesized particles. Our results demonstrate the promise of implementing synthetic gene circuits for tunable control of nanomaterials made by biological systems

A neural network model predicts community-level signaling states in a diverse microbial community.

Signal crosstalk within biological communication networks is common, and such crosstalk can have unexpected consequences for decision making in heterogeneous communities of cells. Here we examined crosstalk within a bacterial community composed of five strains of Bacillus subtilis, with each strain producing a variant of the quorum sensing peptide ComX. In isolation, each strain produced one variant of the ComX signal to induce expression of genes associated with bacterial competence. When strains were combined, a mixture of ComX variants was produced resulting in variable levels of gene expression. To examine gene regulation in mixed communities, we implemented a neural network model. Experimental quantification of asymmetric crosstalk between pairs of strains parametrized the model, enabling the accurate prediction of activity within the full five-strain network. Unlike the single strain system in which quorum sensing activated upon exceeding a threshold concentration of the signal, crosstalk within the five-strain community resulted in multiple community-level quorum sensing states, each with a unique combination of quorum sensing activation among the five strains. Quorum sensing activity of the strains within the community was influenced by the combination and ratio of strains as well as community dynamics. The community-level signaling state was altered through an external signal perturbation, and the output state depended on the timing of the perturbation. Given the ubiquity of signal crosstalk in diverse microbial communities, the application of such neural network models will increase accuracy of predicting activity within microbial consortia and enable new strategies for control and design of bacterial signaling networks

Efflux pump gene amplifications bypass necessity of multiple target mutations for resistance against dual-targeting antibiotic

Abstract Antibiotics that have multiple cellular targets theoretically reduce the frequency of resistance evolution, but adaptive trajectories and resistance mechanisms against such antibiotics are understudied. Here we investigate these in methicillin resistant Staphylococcus aureus (MRSA) using experimental evolution upon exposure to delafloxacin (DLX), a novel fluoroquinolone that targets both DNA gyrase and topoisomerase IV. We show that selection for coding sequence mutations and genomic amplifications of the gene encoding a poorly characterized efflux pump, SdrM, leads to high DLX resistance, circumventing the requirement for mutations in both target enzymes. In the evolved populations, sdrM overexpression due to genomic amplifications containing sdrM and two adjacent genes encoding efflux pumps results in high DLX resistance, while the adjacent hitchhiking efflux pumps contribute to streptomycin cross-resistance. Further, lack of sdrM necessitates mutations in both target enzymes to evolve DLX resistance, and sdrM thus increases the frequency of resistance evolution. Finally, sdrM mutations and amplifications are similarly selected in two diverse clinical isolates, indicating the generality of this DLX resistance mechanism. Our study highlights that instead of reduced rates of resistance, evolution of resistance to multi-targeting antibiotics can involve alternate high-frequency evolutionary paths, that may cause unexpected alterations of the fitness landscape, including antibiotic cross-resistance

Quantifying the strength of quorum sensing crosstalk within microbial communities

<div><p>In multispecies microbial communities, the exchange of signals such as acyl-homoserine lactones (AHL) enables communication within and between species of Gram-negative bacteria. This process, commonly known as quorum sensing, aids in the regulation of genes crucial for the survival of species within heterogeneous populations of microbes. Although signal exchange was studied extensively in well-mixed environments, less is known about the consequences of crosstalk in spatially distributed mixtures of species. Here, signaling dynamics were measured in a spatially distributed system containing multiple strains utilizing homologous signaling systems. Crosstalk between strains containing the <i>lux</i>, <i>las</i> and <i>rhl</i> AHL-receptor circuits was quantified. In a distributed population of microbes, the impact of community composition on spatio-temporal dynamics was characterized and compared to simulation results using a modified reaction-diffusion model. After introducing a single term to account for crosstalk between each pair of signals, the model was able to reproduce the activation patterns observed in experiments. We quantified the robustness of signal propagation in the presence of interacting signals, finding that signaling dynamics are largely robust to interference. The ability of several wild isolates to participate in AHL-mediated signaling was investigated, revealing distinct signatures of crosstalk for each species. Our results present a route to characterize crosstalk between species and predict systems-level signaling dynamics in multispecies communities.</p></div

The weight parameters derived from the comparison of the model simulations and the experimental results.

<p>The weight parameters derived from the comparison of the model simulations and the experimental results.</p

The dependence of activation dynamics on the number of interactor cells.

<p><b>A.</b> The addition of the LasI strain as the interactor strain reduces the activation time compared to case of no crosstalk. <b>B.</b> The addition of the RhlI strain as the interactor increases activation times compared to the no crosstalk case. In both cases, the shift in activation time was proportional to the amount of interactor strain added, as defined by the ratio of interactor to receiver (see text for details). Experimental data are from three independent measurements. The plots in the background show the trend of the data and are solely meant to guide the eye.</p

Testing the model and experiments with natural isolates.

<p><b>A.</b> The comparison of simulation results to the experimental results with <i>Pseudomonas aeruginosa</i> as the interactor strain. Data from the LasI and RhlI interactor strains were used to predict the combined influence of the LasI and RhlI quorum sensing circuits in <i>P</i>. <i>aeruginosa</i>. The LasI + RhlI + growth influences line adds the experimentally measured reduction in the growth rate of <i>E</i>. <i>coli</i> in the presence of <i>P</i>. <i>aeruginosa</i> to the model, see Figures R-T in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005809#pcbi.1005809.s001" target="_blank">S1 File</a>. <b>B.</b> The plate assay was used to measure the interference potential of four wild bacterial isolates at 0.9 ratio of interactor to receiver. Lines, shown to guide the eye, are exponential fits to the data.</p

Robustness of the activation of gene expression to crosstalk.

<p><b>A,B.</b> Theoretical predictions for the response of the receiver cells in the presence of excitatory crosstalk or inhibitory crosstalk. The experimental data from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005809#pcbi.1005809.g003" target="_blank">Fig 3</a> are shown for comparison. <b>C.</b> Comparisons between the experimental measurements of the activation of gene expression in the plate assay to predictions made using the reaction diffusion model. Lines show the predicted change in the activation time at multiple distances from the sender colony as a function of the amount of interactor strain added to the plate. Predictions were made using the experimentally calculated crosstalk weights for the RhlI and LasI interactor strains. Data points show experimental measurements at selected distances from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005809#pcbi.1005809.g003" target="_blank">Fig 3</a>.</p

The bacterial strains used in this study.

<p>The bacterial strains used in this study.</p