Employing aromatic tuning to modulate output from two-component signaling circuits

Abstract

Two-component signaling circuits (TCSs) govern the majority of environmental, pathogenic and industrial processes undertaken by bacteria. Therefore, controlling signal output from these circuits in a stimulus-independent manner is of central importance to synthetic microbiologists. Aromatic tuning, or repositioning the aromatic residues commonly found at the cytoplasmic end of the final TM helix has been shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor (EnvZ) of Escherichia coli. Aromatic residues are found in a similar location within other bacterial membrane-spanning receptors, suggesting that aromatic tuning could be harnessed for a wide-range of applications. Here, a brief synopsis of the data underpinning aromatic tuning, the initial successes with the method and the inherent advantages over those previously employed for modulating TCS signal output are presented.

Keywords: Aromatic tuning; Receptor engineering; Signal modulation; Synthetic circuits; Synthetic microbiology.

Fig. 1

Modularity of two-component signaling circuits (TCSs). When the sensor domain of a canonical SHK perceives stimulus, communication occurs across the membrane (black line) resulting in increased kinase activity of the catalytic ATPase (CA) domain. This enhances phosphorylation of the conserved histidyl residue within the domain responsible for dimerization and histidylphosphotransfer (DHp). These nascent phosphoryl groups are subsequently transferred to an aspartyl residue within the receiver domain of the RR, which usually increases the DNA-binding activity of the output domain leading to transcription of a group of genes, known as a regulon, related to the cognate stimulus [1]. Aromatic tuning, or moving aromatic residues (red box) at the cytoplasmic end of the transmembrane (TM) domain, facilitates stimulus-independent modulation of signaling circuits by mimicking the presence of cognate stimulus and thus altering SHK output, and in turn, transcription of the associated regulon

Fig. 2

Schematic summary of the evidence supporting the affinity of amphipathic aromatic residues for polar/hydrophobic interfaces. a Mixing peptides consisting of a poly-Ala-Leu core of different lengths flanked by Trp residues (WALP peptides) with synthetic bilayers of different thicknesses demonstrates the affinity of the Trp residues for the polar/hydrophobic interfaces [14, 15]. When the distance between the Trp residues was sufficiently shorter than the distance between the polar/hydrophobic interfaces, the lipids adopted an inverted hexagonal (H_II) phase to accommodate interactions between the Trp residues and the interfacial regions (left panel). When the distance between the flanking aromatic residues was increased so that it matched the distance between the interfacial regions, lamellar phases were observed (center panel). As the distance between Trp residues was increased, the acyl chains became more ordered suggesting a slight expansion of the bilayer to accommodate these “larger” peptides (right panel). b Glycosylation-mapping analysis employs a Lep model protein with two TM helices [18]. Segments to be analyzed are inserted at TM2 and a glycosylation-accepting site is positioned between 6 and 11 residues away from the lumenal boundary of TM2. Glycosylation-accepting sites shown in green are distal enough to become glycosylated while those in red are not. Proximity of an accepting site to the active site of oligosaccharyltransferase (OST) correlated with the extent of glycosylation. Therefore, repositioning of TM2 will change these relative positions and hence the extent of glycosylation. c Calculation of minimum glycosylation distance (MGD). MGD simply indicates the number of residues required for half-maximal glycosylation. Repositioning of TM2 into the membrane will increase MGD, while outward displacements of TM2 result in a reduction of MGD. Previous changes in MGD have demonstrated that moving Trp residues about the lumenal end of TM2 resulted in bidirectional, i.e., into and out of the membrane, displacement of the TM helix [19]

Fig. 3

The chemotactic circuit underlying control of flagellar rotation. a In the absence of chemoeffectors, baseline CheA activity maintains phsopho-CheY levels that produce the three-dimensional random walk underlying canonical bacterial chemotaxis. b Binding of attractant (red oval in the periplasm; peri) to the chemoreceptor, i.e., aspartate to Tar, abolishes CheA activity, thereby decreasing intracellular phospho-CheY levels. This also results in reduced methylesterase activity due to reduced CheB-P levels. Transmembrane communication (across the black line) is believed to occur via a piston-type displacement of TM2 toward the cytoplasm (cyto; center panel). c Adaptive methylation (blue dots in the cytoplasm) due to reduced CheB-P levels, restores the ability of the chemoreceptor to stimulate CheA activity when it is occupied by an attractant ligand [23]. In summary, this circuit was an excellent initial target for aromatic tuning because binding of attractant leads to displacement of TM2 toward the cytoplasm, reduced CheA kinase activity and increased levels of covalent modification. Conversely, displacements of TM2 toward the periplasm are consistent with increased CheA activity and reduced levels of covalent modification. In addition, signal output from Tar that is biased beyond the compensatory extent of methylation can be detected by monitoring rotation of individual flagella

Fig. 4

Incremental tuning of signal output from the aspartate chemoreceptor of E. coli (Tar). a At the C-terminal end of Tar TM2, a Trp-Tyr (red) was moved about its original position at the cytoplasmic polar/hydrophobic interface [26]. b The intracellular level of CheY-P governs the probability of clockwise flagellar rotation (P_CW). Previous results demonstrate that increased intracellular CheY-P levels lead to an enhanced probability of CW rotation. In these experiments, a sharp transition was observed, i.e., a Hill coefficient of more than 10 [65]. c Rotation of a single flagellum from roughly 200 independent E. coli cells expressing one of the aromatically tuned variants were analyzed and classified into one of five categories (from left to right): rotating exclusively CCW, rotating primarily CCW with occasional reversals, rapidly switching between both rotational directions (CW/CCW), rotating primarily CW with occasional reversals and those rotating exclusively CW. As Tar signal output increases, the number of cells in each category shifts from CCW toward CW rotational bias. In summary, the lowest overall signal output was observed from cells expressing the WY-3 variant, while the greatest was observed from cells expressing the WY + 2 or WY + 3 variants. Therefore, in the case of Tar, the absolute vertical position of the aromatic residues correlates with signal output [26]

Fig. 5

Non-incremental tuning of signal output from the major osmosensor of E. coli (EnvZ). a EnvZ is bifunctional and possesses both kinase and phosphatase activity. The ratio of these activities is modulated by several factors including the presence of extracellular osmolarity [66, 67], procaine [68] or MzrA [69, 70]. OmpR serves as the cognate RR of EnvZ and the intracellular level of phosphorylated OmpR (OmpR-P) in governed by EnvZ activity. b OmpR-P levels control transcription of ompF and ompC, which can be monitored by employing an E. coli strain that contains a transcriptional fusion of yfp to ompF (yellow) and of cfp to ompC (blue) [7]. Intracellular levels of OmpR-P (red) can thus be estimated by monitoring the CFP/YFP ratio. c Experimental results demonstrating that increasing sucrose levels in the growth medium resulted in increasing levels of CFP, decreasing levels of YFP and an increase in the CFP/YFP ratio. The black dashed line in Fig. 5B represents signal output from this strain upon growth in medium with no additional sucrose. d When aromatic tuning was performed in EnvZ, a Trp-Leu-Phe triplet (red) was repositioned within the C-terminal region of TM2. e The gray-filled circles on the dashed lines indicate the estimated OmpR-P levels in cells expressing one of the aromatically tuned variants. Aromatic tuning in EnvZ resulted in a pattern of signal output that did not correlate with the absolute vertical position of the aromatic residues, as was the case with Tar, but rather appeared approximately helical in distribution suggesting that the surface of TM2 that the residues were located upon was of greater importance [33]. However, the key outcome is that aromatic tuning was still successful with respect to modulating EnvZ signal output in a stimulus-independent manner