Feedback mechanisms are fundamental to the control of physiological responses. One important example in gene regulation, termed negative autoregulation (NAR), occurs when a transcription factor (TF) inhibits its own production through transcriptional repression. This enables more-rapid homeostatic control of gene expression. NAR circuits presumably evolve to limit the fitness costs of gratuitous gene expression. The key biochemical reactions of NAR can be parameterized using a mathematical model of promoter activity; however, this model of NAR has been studied mostly in the context of synthetic NAR circuits that are disconnected from the target genes of the TFs. Thus, it remains unclear how constrained NAR parameters are in a native circuit context, where the TF target genes can have fitness effects on the cell. To quantify these constraints, we created a panel of Escherichia coli strains with different lexA-NAR circuit parameters and analyzed the effect on SOS response function and bacterial fitness. Using a mathematical model for NAR, these experimental data were used to calculate NAR parameter values and derive a parameter-fitness landscape. Without feedback, survival of DNA damage was decreased due to high LexA concentrations and slower SOS “turn-on” kinetics. However, we show that, even in the absence of DNA damage, the lexA promoter is strong enough that, without feedback, high levels of lexA expression result in a fitness cost to the cell. Conversely, hyperfeedback can mimic lexA deletion, which is also costly. This work elucidates the lexA-NAR parameter values capable of balancing the cell’s requirement for rapid SOS response activation with limiting its toxicity. IMPORTANCE Feedback mechanisms are critical to control physiological responses. In gene regulation, one important example, termed negative autoregulation (NAR), occurs when a transcription factor (TF) inhibits its own production. NAR is common across the tree of life, enabling rapid homeostatic control of gene expression. NAR behavior can be described in accordance with its core biochemical parameters, but how constrained these parameters are by evolution is unclear. Here, we describe a model genetic network controlled by an NAR circuit within the bacterium Escherichia coli and elucidate these constraints by experimentally changing a key parameter and measuring its effect on circuit response and fitness. This analysis yielded a parameter-fitness landscape representing the genetic network, providing a window into what gene-environment conditions favor evolution of this regulatory strategy.

Severe infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are often complicated by persistent bacteremia (PB) despite active antibiotic therapy. Antibiotic resistance rarely contributes to MRSA-PB, suggesting an important role for antibiotic tolerance pathways.

Background Persistent MRSA bacteremia is common with high morbidity and mortality despite appropriate antibiotics. Persistent infections are associated with antibiotic tolerance and can arise from perturbations in cellular pathways. We performed whole genome sequencing of clinical isolates to identify the genetic bases of antibiotic tolerance. Methods Whole genomes of MRSA from patients with persistent bacteremia were sequenced, which identified 8 isolates harboring different citZ mutations. To assess the effect of the mutations directly on citrate synthase activity, purified recombinant enzymes were assayed using a commercially available kit. The same kit was used to measure activity in whole cell lysates of MRSA isolates harboring wild-type and mutant citZ alleles. Enzyme kinetic parameters were determined by fitting initial rate data to the Michaelis-Menten equation using nonlinear regression. Figure 1. Whole Genome Sequencing of Persistent MRSA Bacteremia Whole genome sequencing of 206 blood cultures from 20 patients with persistent MRSA bacteremia (defined as >7 days) under the hypothesis that continuous antibiotic exposure will give rise to - and enrich for - mutations that convey antibiotic tolerance through in-host evolution. Results Analysis of whole genomes from 206 blood cultures from 20 patients with persistent MRSA bacteremia identified citZ, which encodes for citrate synthase, the first step of the tricarboxylic acid cycle, as the most repeatedly mutated gene. The data revealed parallelism in its evolution, as citZ was mutated 8 independent times, a rate far greater than from chance alone. To characterize the impact of the mutations on enzyme activity, recombinant proteins were expressed in E. coli and purified for enzymatic assays. As compared to wildtype enzyme, two mutants had reduced activity, and four mutants had near-absent activity; two mutants were unable to be expressed due to destabilizing mutations. Michaelis-Menten analysis of the two mutants with residual activity show that, as compared to wildtype, both had lower affinity for its substrates, and lower maximum activity. Furthermore, we found the cell was unable to compensate for the loss of citrate synthase activity, as lysates harboring these mutations also displayed analogous reductions in activity. Figure 2. Purified citrate synthase mutant proteins have reduced enzyme activity Citrate synthase catalyzes the reaction between acetyl-CoA and oxaloacetic acid to form citric acid with CoA-SH as a byproduct. CoA-SH can interact with DTNB to form TNB, which can be measured spectrophotometrically at A412nm to indirectly measure the activity of citrate synthase. Wildtype citrate synthase and the D141N mutant have comparable activity whereas the remaining study mutants have either reduced (mutants A313P and A313V) or near-absent (mutants G7D, G7D + D141N, S201P, P354S) activity. The three active site mutants (H248G, D309G, H219G) also have near-absent activity and serve as negative controls. Positive control is citrate synthase provided in the Sigma Citrate Synthase Assay Kit. No enzyme control replaces enzyme with assay buffer. All error bars are ± 1 SD, calculated from at least three replicate experiments. Figure 3. A313P and A313V mutants have decreased substrate affinity and decreased maximal activity Panels A and B show plots of the initial rate of citrate synthase activity (Y-axis, in units of µmole/mL/sec) versus OAA/AcCoA substrate concentration (X-axis, in millimolar units), for wildtype citrate synthase and the A313P and A313V mutants. Panel (A) shows enzymatic parameters for wildtype, A313P mutant, and A313V mutant with variable OAA (0-0.625 mM) and fixed AcCoA (0.3 mM). Panel (B) shows enzymatic parameters for wildtype, A313P mutant, and A313V mutant with fixed OAA (0.5 mM) and variable AcCoA (0-2.5 mM). Three replicates were performed for each condition, with error bars showing ± 1 SD. Figure 4. Citrate synthase mutants disrupt functional dimerization and destabilize alpha-helix packing Identified citrate synthase mutants mapped onto the crystal structure of T. thermophilus citrate synthase (PDB 1IOM) show that the mutations are located either at dimerization interfaces or within the interior of hydrophobic packing interfaces. Conclusion Cellular metabolism and virulence regulation are interconnected in S. aureus, as alterations in TCA cycle activity lead to increased persister formation and host macrophage inactivation. Our findings that inactivating citZ mutations are enriched can provide a potential explanation for the mechanism of persistent bacteremia. Disclosures All Authors: No reported disclosures