Taliman Afroz

Bacterial sugar utilization gives rise to distinct single-cell behaviours

Inducible utilization pathways reflect widespread microbial strategies to uptake and consume sugars from the environment. Despite their broad importance and extensive characterization, little is known how these pathways naturally respond to their inducing sugar in individual cells. Here, we performed single‐cell analyses to probe the behaviour of representative pathways in the model bacterium Escherichia coli. We observed diverse single‐cell behaviours, including uniform responses (d‐lactose, d‐galactose, N‐acetylglucosamine, N‐acetylneuraminic acid), ‘all‐or‐none’ responses (d‐xylose, l‐rhamnose) and complex combinations thereof (l‐arabinose, d‐gluconate). Mathematical modelling and probing of genetically modified pathways revealed that the simple framework underlying these pathways – inducible transport and inducible catabolism – could give rise to most of these behaviours. Sugar catabolism was also an important feature, as disruption of catabolism eliminated tunable induction as well as enhanced memory of previous conditions. For instance, disruption of catabolism in pathways that respond to endogenously synthesized sugars led to full pathway induction even in the absence of exogenous sugar. Our findings demonstrate the remarkable flexibility of this simple biological framework, with direct implications for environmental adaptation and the engineering of synthetic utilization pathways as titratable expression systems and for metabolic engineering.

Trade-offs in Engineering Sugar Utilization Pathways for Titratable Control

Titratable systems are common tools in metabolic engineering to tune the levels of enzymes and cellular components as part of pathway optimization. For nonmodel microorganisms with limited genetic tools, inducible sugar utilization pathways offer built-in titratable systems. However, these pathways can exhibit undesirable single-cell behaviors that hamper the uniform and tunable control of gene expression. Here, we applied mathematical modeling and single-cell measurements of l-arabinose utilization in Escherichia coli to systematically explore how sugar utilization pathways can be altered to achieve desirable inducible properties. We found that different pathway alterations, such as the removal of catabolism, constitutive expression of high-affinity or low-affinity transporters, or further deletion of the other transporters, came with trade-offs specific to each alteration. For instance, sugar catabolism improved the uniformity and linearity of the response at the cost of requiring higher sugar concentrations to induce the pathway. Within these alterations, we also found that a uniform and linear response could be achieved with a single alteration: constitutively expressing the high-affinity transporter. Equivalent modifications to the d-xylose utilization pathway yielded similar responses, demonstrating the applicability of our observations. Overall, our findings indicate that there is no ideal set of typical alterations when co-opting natural utilization pathways for titratable control and suggest design rules for manipulating these pathways to advance basic genetic studies and the metabolic engineering of microorganisms for optimized chemical production.

Rethinking the Hierarchy of Sugar Utilization in Bacteria.

Bacteria are known to consume some sugars over others, although recent work reported by Koirala and colleagues in this issue of the Journal of Bacteriology (S. Koirala, X. Wang, and C. V. Rao, J Bacteriol 198:386-393, 2016, http://dx.doi.org/10.1128/JB.00709-15) revealed that individual cells do not necessarily follow this hierarchy. By studying the preferential consumption of l-arabinose over d-xylose in Escherichia coli, those authors found that subpopulations consume one, the other, or both sugars through cross-repression between utilization pathways. Their findings challenge classic assertions about established hierarchies and can guide efforts to engineer the simultaneous utilization of multiple sugars.

Impact of Residual Inducer on Titratable Expression Systems

Inducible expression systems are widely employed for the titratable control of gene expression, yet molecules inadvertently present in the growth medium or synthesized by the host cells can alter the response profile of some of these systems. Here, we explored the quantitative impact of these residual inducers on the apparent response properties of inducible systems. Using a simple mathematical model, we found that the presence of residual inducer shrinks the apparent dynamic range and causes the apparent Hill coefficient to converge to one. We also found that activating systems were more sensitive than repressing systems to the presence of residual inducer and the response parameters were most heavily dependent on the original Hill coefficient. Experimental interrogation of common titratable systems based on an L-arabinose inducible promoter or a thiamine pyrophosphate-repressing riboswitch in Escherichia coli confirmed the predicted trends. We finally found that residual inducer had a distinct effect on “all-or-none” systems, which exhibited increased sensitivity to the added inducer until becoming fully induced. Our findings indicate that residual inducer or repressor alters the quantitative response properties of titratable systems, impacting their utility for scientific discovery and pathway engineering.

A "Looking Glass" into Electrolyte Properties: Cyclic Carbonate and Ester-LiClO4 Mixtures

Many electrolyte mixtures have been introduced and tested for Li-ion batteries. Most of this work has been done by a trial-and-error approach. In part, this is due to the current limited understanding regarding electrolyte interactions at the molecular level—specifically ionic association and solvation. The properties of electrolytes are dictated by these molecular interactions, which are influenced by several factors, such as the structure of the anion and solvent, temperature and salt concentration. This study focuses on LiClO4 mixtures with different cyclic carbonate and ester solvents: ethylene carbonate (EC), propylene carbonate (PC), γ-butyrolactone (GBL), and γ-valerolactone (GVL). LiClO4, like LiBF4, is an intermediately associated salt and has been used for electrolytes in primary lithium batteries. Carbonate solvents, especially EC, are the most widely used solvents for electrolytes. To understand how the interactions are influenced by differences in solvent structure, the solventLiClO4 mixtures have been examined with thermal and vibrational spectroscopic analysis. From DSC measurements, phase diagrams have been prepared (i.e., Fig. 1). Crystalline solvate phases have been identified and the structures of these solvates obtained by single crystal XRD analysis. These solvates show the coordination between the ions and solvent in the crystalline phases. In addition, Raman spectroscopic analysis has been used to examine the solvent and anion interactions in both the solid-state and liquid phases. This information provides insight into the types of solvates and their relative amounts present in the liquid electrolytes. This then enables the direct correlation between the molecular-level interactions and electrolyte physical properties such as ionic conductivity, viscosity, wettability, volatility, etc. Acknowledgement: This research was fully supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award ER46655.