Modeling Chemotaxis Reveals the Role of Reversed Phosphotransfer and a Bi-Functional Kinase-Phosphatase

Understanding how multiple signals are integrated in living cells to produce a balanced response is a major challenge in biology. Two-component signal transduction pathways, such as bacterial chemotaxis, comprise histidine protein kinases (HPKs) and response regulators (RRs). These are used to sense and respond to changes in the environment. Rhodobacter sphaeroides has a complex chemosensory network with two signaling clusters, each containing a HPK, CheA. Here we demonstrate, using a mathematical model, how the outputs of the two signaling clusters may be integrated. We use our mathematical model supported by experimental data to predict that: (1) the main RR controlling flagellar rotation, CheY₆, aided by its specific phosphatase, the bifunctional kinase CheA₃, acts as a phosphate sink for the other RRs; and (2) a phosphorelay pathway involving CheB₂ connects the cytoplasmic cluster kinase CheA₃ with the polar localised kinase CheA₂, and allows CheA₃-P to phosphorylate non-cognate chemotaxis RRs. These two mechanisms enable the bifunctional kinase/phosphatase activity of CheA₃ to integrate and tune the sensory output of each signaling cluster to produce a balanced response. The signal integration mechanisms identified here may be widely used by other bacteria, since like R. sphaeroides, over 50% of chemotactic bacteria have multiple cheA homologues and need to integrate signals from different sources.     

Interaction Analysis of a Two-Component System Using Nanodiscs

Two-component systems are the major means by which bacteria couple adaptation to environmental changes. All utilize a phosphorylation cascade from a histidine kinase to a response regulator, and some also employ an accessory protein. The system-wide signaling fidelity of two-component systems is based on preferential binding between the signaling proteins. However, information on the interaction kinetics between membrane embedded histidine kinase and its partner proteins is lacking. Here, we report the first analysis of the interactions between the full-length membrane-bound histidine kinase CpxA, which was reconstituted in nanodiscs, and its cognate response regulator CpxR and accessory protein CpxP. Using surface plasmon resonance spectroscopy in combination with interaction map analysis, the affinity of membrane-embedded CpxA for CpxR was quantified, and found to increase by tenfold in the presence of ATP, suggesting that a considerable portion of phosphorylated CpxR might be stably associated with CpxA in vivo. Using microscale thermophoresis, the affinity between CpxA in nanodiscs and CpxP was determined to be substantially lower than that between CpxA and CpxR. Taken together, the quantitative interaction data extend our understanding of the signal transduction mechanism used by two-component systems.     

Using Information Interaction to Discover Epistatic Effects in Complex Diseases

It is widely agreed that complex diseases are typically caused by the joint effects of multiple instead of a single genetic variation. These genetic variations may show stronger effects when considered together than when considered individually, a phenomenon known as epistasis or multilocus interaction. In this work, we explore the applicability of information interaction to discover pairwise epistatic effects related with complex diseases. We start by showing that traditional approaches such as classification methods or greedy feature selection methods (such as the Fleuret method) do not perform well on this problem. We then compare our information interaction method with BEAM and SNPHarvester in artificial datasets simulating epistatic interactions and show that our method is more powerful to detect pairwise epistatic interactions than its competitors. We show results of the application of information interaction method to the WTCCC breast cancer dataset. Our results are validated using permutation tests. We were able to find 89 statistically significant pairwise interactions with a p-value lower than . Even though many recent algorithms have been designed to find epistasis with low marginals, we observed that all (except one) of the SNPs involved in statistically significant interactions have moderate or high marginals. We also report that the interactions found in this work were not present in gene-gene interaction network STRING.     

Use of a Probabilistic Motif Search to Identify Histidine Phosphotransfer Domain-Containing Proteins

The wealth of newly obtained proteomic information affords researchers the possibility of searching for proteins of a given structure or function. Here we describe a general method for the detection of a protein domain of interest in any species for which a complete proteome exists. In particular, we apply this approach to identify histidine phosphotransfer (HPt) domain-containing proteins across a range of eukaryotic species. From the sequences of known HPt domains, we created an amino acid occurrence matrix which we then used to define a conserved, probabilistic motif. Examination of various organisms either known to contain (plant and fungal species) or believed to lack (mammals) HPt domains established criteria by which new HPt candidates were identified and ranked. Search results using a probabilistic motif matrix compare favorably with data to be found in several commonly used protein structure/function databases: our method identified all known HPt proteins in the Arabidopsis thaliana proteome, confirmed the absence of such motifs in mice and humans, and suggests new candidate HPts in several organisms. Moreover, probabilistic motif searching can be applied more generally, in a manner both readily customized and computationally compact, to other protein domains; this utility is demonstrated by our identification of histones in a range of eukaryotic organisms.     

Structural Basis for the Localization of the Chemotaxis Phosphatase CheZ by CheAS

CheA-short interacts with CheZ to localize CheZ to cell poles. The fifth helical region (residues 112 to 133) from the phosphotransfer domain of CheA interacts with CheZ and becomes ordered and helical, although it lacks a stable fold in the CheA fragment comprising residues 98 to 150 alone. One CheA molecule binds to one CheZ dimer.During bacterial chemotaxis, transmembrane receptors regulate the activity of the chemotaxis-specific histidine autokinase CheA with the aid of a coupling protein, CheW. CheA acts to phosphorylate the response regulator CheY and the response regulator domain of the methylesterase CheB. Phosphorylated CheY (CheY-P) binds to the “switch complex” in the flagellar motor to regulate the sense of rotation of the motor. CheZ acts as a CheY phosphate phosphatase.Maddock and Shapiro (4) showed that the chemotaxis receptors tend to be clustered and often located at polar ends of bacterial cells. This localization of receptors is in large part dependent on the presence of CheA and CheW, and the clusters that form in wild-type cells contain receptors, CheA, CheW, CheY, and CheZ (8). These clusters are essential for proper communication among receptors and other members of the signal transduction complex.In Escherichia coli and many related bacteria, a naturally occurring short form of CheA (CheA_S) (7) interacts with CheZ, enhances the rate of dephosphorylation of CheY-P (5, 10), and is responsible for the localization of CheZ to the polar assemblies of receptors, CheA, and CheW (1). Having the kinase and the phosphatase colocalized generates more uniform CheY levels within the bacterial cell (9).In order to understand the structural basis of the CheA_S-CheZ interaction, we examined a CheA fragment containing residues 98 to 150 (CheA_98-150). This fragment begins at the alternative site of translation initiation for CheA_S and extends into the linker region joining the histidine phosphotransfer domain to the CheY-binding domain. This fragment includes residues that correspond to the C terminus of the fourth helix and the complete fifth helix of the intact histidine phosphotransfer domain, also known as the P1 domain. Figure ​Figure11 depicts the binding of CheA_98-150 to CheZ, detected by changes in the fluorescence of the tryptophan residues of CheZ. The data points represent the fluorescence intensities from the complex, plotted against CheA_98-150 concentrations. Assay results were collected in triplicate, and the data points indicate the mean values. The fluorescence intensity (excitation wavelength, 295 nm; emission wavelength, 340 nm) was monitored after each addition and corrected for the blank buffer. The solid lines represent least-squares fits to a two-state binding model. As shown in Fig. ​Fig.1,1, CheA_98-150 binds to CheZ with a dissociation constant in the nanomolar range, with a stoichiometry of one CheA_98-150 molecule per CheZ dimer.Open in a separate windowFIG. 1.Intrinsic tryptophan fluorescence detection of the interactions of wild-type CheZ (A) and CheZ_65-139 (B) with CheA_98-150. Wild-type CheZ and CheZ_65-139 (both present at 1.0 μM) were titrated with CheA_98-150 to produce saturation binding curves at 25°C. The dissociation constants fit to values between 10 and 30 nM but are too strong to be determined accurately at these CheZ concentrations. arb units, arbitrary units.Figure ​Figure22 shows the ¹H-¹⁵N correlation spectrum for CheA_98-150. The resonances were mostly resolved and sharp, with a limited dispersion of chemical shifts along the ¹H dimension, suggesting a high degree of backbone mobility (2). Complete backbone assignments for the nonproline residues in CheA_98-150 were made using standard HNCACB and CBCA(CO)NH methods (6). We have assigned Glu100 through His154 (a residue of the His₆ tag). Although the nuclear magnetic resonance (NMR) spectra and ¹⁵N relaxation properties (3) suggest that this fragment has no stable structure under these conditions, the central region (Asp112 to Glu133) exhibits positive (¹H-)¹⁵N nuclear Overhauser effects and large, positive (up to 2.6 ppm) Cα secondary shifts (11), consistent with a partially rigid, helical structure.Open in a separate windowFIG. 2.¹H-¹⁵N heteronuclear single-quantum coherence spectra of 100 mM ¹⁵N-labeled CheA_98-150 in the absence (black) and presence (red) of 200 mM unlabeled CheZ. Amino acid residues disappearing (black) or displaying significant chemical-shift perturbations (red) are identified.To establish which residues of CheA_98-150 are involved in CheZ binding, we titrated ¹⁵N-labeled CheA_98-150 with unlabeled wild-type CheZ. The spectra were collected using a mixture of 50 mM sodium phosphate buffer, pH 6.8, 1 mM EDTA, and 2 mM dithiothreitol at 25°C. The ¹H-¹⁵N resonances from Asp112 to Glu133, the fifth helix of the N-terminal domain in CheA, weakened as CheZ was added, and these peaks disappeared with the addition of two molar equivalents of CheZ subunits. A new set of resonances, albeit somewhat broadened, appeared near Asp112 to Glu133, suggesting that these residues are at the periphery of the binding region and retain sufficient mobility in the bound state to be observed. New resonances for these residues from the bound state strengthened, whereas resonances from the free state weakened, as the CheZ concentration increased. This pattern of resonance changes is characteristic of an interaction that is in the slow-exchange regime on the NMR time scale. The disappearance of the CheA resonances at a 1:2 ratio with CheZ again indicates that the binding stoichiometry is two CheZ monomers per CheA_98-150 molecule, and the bound complex in the protonated form is likely too large (∼100 kDa) or elongated to be observed.To examine the CheA-CheZ interaction in more detail, we assigned the amide resonances from CheA_98-150 in a complex with a shortened form of CheZ comprising residues 65 to 139 (CheZ_65-139). We chose to focus on this central region of CheZ because it gave sharper CheA resonances in the complex but still bound CheA_98-150 very strongly. The ¹³C backbone chemical shifts of the bound form of CheA_98-150 support a fully helical structure (12) for the region of Asp 112 to Glu133.Results from chemical shift perturbation experiments with CheZ_65-139 indicate that the CheA_98-150-binding site in CheZ is the helix bundle tip, where several aromatic residues cluster (data not shown). Several extreme-upfield methyl proton resonances of CheZ_65-139 shifted downfield upon binding with CheA_98-150, indicating a reorganization of aliphatic methyl groups and aromatic rings in CheZ. Many CheZ_65-139 resonances shifted upon binding, strongly indicating that CheA_S induces global structural changes that propagate from the binding site toward the central, CheY-binding region. This possibility is consistent with the observed perturbations of histidine side chain resonances of CheZ_65-139 at the other end of the helix bundle (data not shown). The imidazole rings from the four histidines in a CheZ_65-139 dimer are situated 25 to 30 Å from the helix bundle tip. The resonances from the imidazole nitrogen atoms and carbon-bound protons detected by ¹H-¹⁵N correlation spectra are clearly affected by the binding of CheA_98-150.We identified the region from Asp112 to Glu133 in CheA_98-150 as being responsible for CheZ binding. This region corresponds to the fifth helix in the intact P1 domain of CheA. NMR data indicate that this region is still mildly helical in CheA_98-150, although it lacks folding cooperativity. The helical content is enhanced by CheZ binding. A model of the complex with one CheA and two CheZ molecules was built (Fig. ​(Fig.3).3). The CheA_S helix formed by residues 112 to 133 is shown bound to an opening formed by the two helical hairpins in a CheZ dimer. A space-filling model (not shown) indicates that there is not enough room to accommodate the CheA helix, suggesting that near the hairpin turn region the four-helix bundle of CheZ expands upon the binding of CheA_S, resulting in structural changes remote from the binding area. This assumption is consistent with the extensive peak movements observed in the CheZ_65-139 spectrum upon the binding of CheA_S. These binding-induced structural changes near the middle of the CheZ helical bundle are likely to be responsible for enhanced CheY-P-binding affinity and/or catalysis of phosphate hydrolysis, leading to increased CheY-P phosphatase activity.Open in a separate windowFIG. 3.Model of the CheZ-CheA_S interaction. The model shows residues 65 to 139 of CheZ as a helical ribbon (Protein Data Bank identification number 1KMI). The cluster of aromatic residues in CheZ is shown in magenta, and the helical CheA residues 112 to 133 are shown end-on with blue and red side chains. The CheA helix (residues 112 to 133) was taken from the known structure of the Htp domain of CheA (Protein Data Bank identification number 1I5N) and manually docked with CheZ to maximize hydrophobic contact.     

Structural Specificity of Sugar Transport at the Blood-Nerve Barrier

Abstract: The major component of D-glucose transfer across the membranous sites of the blood-nerve barrier (BNB) occurs via a facilitative mechanism at a rate greater than twice the rate of D-glucose metabolism by nerve. To characterize further properties of monosaccharide transport at the BNB, unidirectional transfer constant (K) values were determined in vivo in tibial nerve of anesthetized rats for radiolabeled mannitol, L-glucose, and a series of D-glucose analogs. K values (× 10⁻⁴ ml s⁻¹ g^−l) equaled 4.8 for 2-deoxy-D-glucose, 3.7 for D-glucose, 2.3 for 3- O -methyl-D-glucose, 1.4 for D-man-nose, 0.6 for D-galactose, 0.2 for mannitol, and 0.19 for L-glucose. The rank order of ratios between K values of a D-hexose and D-glucose, which reflects the rank order of affinity of the system for individual sugars, was 2-deoxy-D-glucose > D-glucose > 3-O-methyl-D-glucose > D-mannose > D-galactose. The results demonstrate that the order of substrate affinity of the monosaccharide carrier at the BNB is similar to that at cerebral capillaries and at erythrocytes. At normal concentrations of plasma D-glucose, the contribution of simple passive diffusion to unidirectional D-glucose influx across the BNB equals 5%, which is greater than that at cerebral capillaries and reflects the greater permeability to hydrophilic nonelectrolytes of the endoneurial vasculature.     

Vertical Signalling Involves Transmission of Hox Information from Gastrula Mesoderm to Neurectoderm

Development and patterning of neural tissue in the vertebrate embryo involves a set of molecules and processes whose relationships are not fully understood. Classical embryology revealed a remarkable phenomenon known as vertical signalling, a gastrulation stage mechanism that copies anterior-posterior positional information from mesoderm to prospective neural tissue. Vertical signalling mediates unambiguous copying of complex information from one tissue layer to another. In this study, we report an investigation of this process in recombinates of mesoderm and ectoderm from gastrulae of Xenopus laevis. Our results show that copying of positional information involves non cell autonomous autoregulation of particular Hox genes whose expression is copied from mesoderm to neurectoderm in the gastrula. Furthermore, this information sharing mechanism involves unconventional translocation of the homeoproteins themselves. This conserved primitive mechanism has been known for three decades but has only recently been put into any developmental context. It provides a simple, robust way to pattern the neurectoderm using the Hox pattern already present in the mesoderm during gastrulation. We suggest that this mechanism was selected during evolution to enable unambiguous copying of rather complex information from cell to cell and that it is a key part of the original ancestral mechanism mediating axial patterning by the highly conserved Hox genes.     

Screening of Marine Bacteria for the Production of Microbial Repellents Using a Spectrophotometric Chemotaxis Assay

A method for screening marine bacteria for the production of microbial repellents has been developed. The spectrophotometer provided quantitative information on bacterial chemotaxis in response to extracts from other strains of marine bacteria. Aqueous extracts were incorporated into an agar plug at the base of a cuvette, which was overlaid with a suspension of a motile strain. Negative chemotaxis of the motile strain in response to diffusion of repellent compounds from the agar could be measured by a fall in the optical density, allowing the direct screening of supernatants for repellent activity. Three strains producing metabolites with a repellent effect on a motile marine bacterium were identified. Antibiotic activity and the repellent effect of the supernatants were compared, with no significant correlation being found. The screening method will therefore allow the identification of bioactive metabolites that would be overlooked using traditional antibiotic screening strategies. Received March 4, 1998; accepted November 11, 1998.     

Identification of a Novel Staphylococcus aureus Two-Component Leukotoxin Using Cell Surface Proteomics

Staphylococcus aureus is a prominent human pathogen and leading cause of bacterial infection in hospitals and the community. Community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 are highly virulent and, unlike hospital strains, often cause disease in otherwise healthy individuals. The enhanced virulence of CA-MRSA is based in part on increased ability to produce high levels of secreted molecules that facilitate evasion of the innate immune response. Although progress has been made, the factors that contribute to CA-MRSA virulence are incompletely defined. We analyzed the cell surface proteome (surfome) of USA300 strain LAC to better understand extracellular factors that contribute to the enhanced virulence phenotype. A total of 113 identified proteins were associated with the surface of USA300 during the late-exponential phase of growth in vitro. Protein A was the most abundant surface molecule of USA300, as indicated by combined Mascot score following analysis of peptides by tandem mass spectrometry. Unexpectedly, we identified a previously uncharacterized two-component leukotoxin–herein named LukS-H and LukF-G (LukGH)-as two of the most abundant surface-associated proteins of USA300. Rabbit antibody specific for LukG indicated it was also freely secreted by USA300 into culture media. We used wild-type and isogenic lukGH deletion strains of USA300 in combination with human PMN pore formation and lysis assays to identify this molecule as a leukotoxin. Moreover, LukGH synergized with PVL to enhance lysis of human PMNs in vitro, and contributed to lysis of PMNs after phagocytosis. We conclude LukGH is a novel two-component leukotoxin with cytolytic activity toward neutrophils, and thus potentially contributes to S. aureus virulence.     

Structural Basis of the pH-Dependent Assembly of a Botulinum Neurotoxin Complex

Botulinum neurotoxins (BoNTs) are among the most poisonous biological substances known. They assemble with non-toxic non-hemagglutinin (NTNHA) protein to form the minimally functional progenitor toxin complexes (M-PTC), which protects BoNT in the gastrointestinal tract and releases it upon entry into the circulation. Here we provide molecular insight into the assembly between BoNT/A and NTNHA-A using small-angle X-ray scattering. We found that the free form BoNT/A maintains a pH-independent conformation with limited domain flexibility. Intriguingly, the free form NTNHA-A adopts pH-dependent conformational changes due to a torsional motion of its C-terminal domain. Once forming a complex at acidic pH, they each adopt a stable conformation that is similar to that observed in the crystal structure of the M-PTC. Our results suggest that assembly of the M-PTC depends on the environmental pH and that the complex form of BoNT/A is induced by interacting with NTNHA-A at acidic pH.     

Chemotaxis in Vitro: Quantitation of Human Granulocyte Movement Using a Stochastic Differential Equation

The quantitation of human granulocyte movement using a stochastic differential equation is described. The method has the potential to distinguish both positive and negative chemotaxis. Analysis and information concerning cell movements can be obtained for any point in time and distance for the duration of the experiment.     

Structural Basis for the SUMO2 Isoform Specificity of SENP7

SUMO proteases or deSUMOylases regulate the lifetime of SUMO-conjugated targets in the cell by cleaving off the isopetidic bond between the substrate and the SUMO modifier, thus reversing the conjugation activity of the SUMO E3 ligases. In humans the deSUMOylating activity is mainly conducted by the SENP/ULP protease family, which is constituted of six members sharing a homologous catalytic globular domain. SENP6 and SENP7 are the most divergent members of the family and they show a unique SUMO2/3 isoform preference and a particular activity for dismantling polySUMO2 chains. Here, we present the crystal structure of the catalytic domain of human SENP7 bound to SUMO2, revealing structural key elements for the SUMO2 isoform specificity of SENP7. In particular, we describe the specific contacts between SUMO2 and a unique insertion in SENP7 (named Loop1) that is responsible for the SUMO2 isoform specificity. All the other interface contacts between SENP7 and SUMO2, including the SUMO2 C-terminal tail interaction, are conserved among members of the SENP/ULP family. Our data give insight into an evolutionary adaptation to restrict the deSUMOylating activity in SENP6 and SENP7 for the SUMO2/3 isoforms.     

Structural Specificity of Polyamines in Modulating the Binding of Estrogen Receptor to Potential Z-DNA Forming Sequences

Abstract

Estrogen receptor (ER) is a gene-regulatory protein that mediates the action of estradiol. In order to examine the role of conformational dynamics of DNA in estrogenic regulation of gene expression, we studied the binding of ER to poly(dA-dC).poly(dG-dT) which undergoes transition to a left-handed Z-DNA form. This type of dinucleotide repeats are widely distributed in mammalian genome and are present in estrogen response elements. Binding affinity of ER for the polynucleotide was assessed by its ability to release ER bound to DNA-cellulose. ER binding by poly(dA-dC).poly(dG-dT) was enhanced in the presence of an endogenous polyamine, spermidine, H₂N(CH₂)₄NH(CH₂)₃NH₂. The concentration of spermidine required for facilitating 50% elution of ER (EC₅₀) was 75 μM. This EC₅₀ increased to 500 μM for a spermidine homolog, H₂N(CH₂)₈NH(CH₂)₃NH₂, demonstrating polyamine structural specificity. Spectroscopic measurements showed that the presence of 100 – 200 μM spermidine initiated changes in the conformation of the polynucleotide indicative of Z-DNA form, but a major alteration to Z-DNA spectrum occurred only at 300 μM concentration. These data suggest that ER favors DNA sequences poised for Z-DNA transition. The efficacy of spermidine homologs in facilitating ER-DNA interaction may be important in predicting their efficiency to replace cellular functions of spermidine.     

Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity

The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu²⁺ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation.     

Structural Basis for the dsRNA Specificity of the Lassa Virus NP Exonuclease

Lassa virus causes hemorrhagic fever characterized by immunosuppression. The nucleoprotein of Lassa virus, termed NP, binds the viral genome. It also has an additional enzymatic activity as an exonuclease that specifically digests double-stranded RNA (dsRNA). dsRNA is a strong signal to the innate immune system of viral infection. Digestion of dsRNA by the NP exonuclease activity appears to cause suppression of innate immune signaling in the infected cell. Although the fold of the NP enzyme is conserved and the active site completely conserved with other exonucleases in its DEDDh family, NP is atypical among exonucleases in its preference for dsRNA and its strict specificity for one substrate. Here, we present the crystal structure of Lassa virus NP in complex with dsRNA. We find that unlike the exonuclease in Klenow fragment, the double-stranded nucleic acid in complex with Lassa NP remains base-paired instead of splitting, and that binding of the paired complementary strand is achieved by "relocation" of a basic loop motif from its typical exonuclease position. Further, we find that just one single glycine that contacts the substrate strand and one single tyrosine that stacks with a base of the complementary, non-substrate strand are responsible for the unique substrate specificity. This work thus provides templates for development of antiviral drugs that would be specific for viral, rather than host exonucleases of similar fold and active site, and illustrates how a very few amino acid changes confer alternate specificity and biological phenotype to an enzyme.     

Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System

Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli’s natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or “coliroids,” rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein’s local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system’s performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling.