Recently, a synthetic circuit in E. coli demonstrated that two proteins engineered with LAA tags targeted to the native protease ClpXP are susceptible to crosstalk due to competition for degradation between proteins. To understand proteolytic crosstalk beyond the single protease regime, we investigated in E. coli a set of synthetic circuits designed to probe the dynamics of existing and novel degradation tags fused to fluorescent proteins. These circuits were tested using both microplate reader and single-cell assays. We first quantified the degradation rates of each tag in isolation. We then tested if there was crosstalk between two distinguishable fluorescent proteins engineered with identical or different degradation tags. We demonstrated that proteolytic crosstalk was indeed not limited to the LAA degradation tag, but was also apparent between other diverse tags, supporting the complexity of the E. coli protein degradation system.

Modulation of biological oscillations by stimuli lies at the root of many phenomena, including maintenance of circadian rhythms, propagation of neural signals, and somitogenesis. While it is well established that regular periodic modulation can entrain an oscillator, an aperiodic (noisy) modulation can also robustly entrain oscillations. This latter scenario may describe, for instance, the effect of irregular weather patterns on circadian rhythms, or why irregular neural stimuli can still reliably transmit information. A synthetic gene oscillator approach has already proven to be useful in understanding the entrainment of biological oscillators by periodic signaling, mimicking the entrainment of a number of noisy oscillating systems. We similarly seek to use synthetic biology as a platform to understand how aperiodic signals can strongly correlate the behavior of cells. This study should lead to a deeper understanding of how fluctuations in our environment and even within our body may promote substantial synchrony among our cells. Specifically, we investigate experimentally and theoretically the entrainment of a synthetic gene oscillator in E. coli by a noisy stimulus. This phenomenon was experimentally studied and verified by a combination of microfluidics and microscopy using the real synthetic circuit. Stochastic simulation of an associated model further supports that the synthetic gene oscillator can be strongly entrained by aperiodic signals, especially telegraph noise. Finally, widespread applicability of aperiodic entrainment beyond the synthetic gene oscillator is supported by results derived from both a model for a natural oscillator in D. discoideum and a model for predator-prey oscillations.

Toxin–antitoxin (TA) systems are key regulators of bacterial persistence, a multidrug-tolerant state found in bacterial species that is a major contributing factor to the growing human health crisis of antibiotic resistance. Type II TA systems consist of two proteins, a toxin and an antitoxin; the toxin is neutralized when they form a complex. The ratio of antitoxin to toxin is significantly greater than 1.0 in the susceptible population (non-persister state), but this ratio is expected to become smaller during persistence. Analysis of multiple datasets (RNA-seq, ribosome profiling) and results from translation initiation rate calculators reveal multiple mechanisms that ensure a high antitoxin-to-toxin ratio in the non-persister state. The regulation mechanisms include both translational and transcriptional regulation. We classified E. coli type II TA systems into four distinct classes based on the mechanism of differential protein production between toxin and antitoxin. We find that the most common regulation mechanism is translational regulation. This classification scheme further refines our understanding of one of the fundamental mechanisms underlying bacterial persistence, especially regarding maintenance of the antitoxin-to-toxin ratio.

The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants.

Antibiotic tolerance is a widespread phenomenon that renders antibiotic treatments less effective and facilitates antibiotic resistance. Here we explore the role of proteases in antibiotic tolerance, short-term population survival of antibiotics, using queueing theory (i.e. the study of waiting lines), computational models, and a synthetic biology approach. Proteases are key cellular components that degrade proteins and play an important role in a multi-drug tolerant subpopulation of cells, called persisters. We found that queueing at the protease ClpXP increases antibiotic tolerance ~80 and ~60 fold in an E. coli population treated with ampicillin and ciprofloxacin, respectively. There does not appear to be an effect on antibiotic persistence, which we distinguish from tolerance based on population decay. These results demonstrate that proteolytic queueing is a practical method to probe bacterial tolerance and related genes, while limiting the unintended consequences frequently caused by gene knockout and overexpression.