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

Abstract

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.

Fig 1. Phosphotransfers between the two-component signaling proteins in the bacterial photography system.

The EnvZ-OmpR signaling system was modified for bacterial photography system with a light sensing protein introduced as a Cph1-EnvZ fusion protein called Cph8. The absence of light stimulates the autophosphorylation activity Cph8 protein. Cph8 transfers its phosphate to OmpR, increasing the affinity of OmpR for the PompC promoter and activating transcription of LacZ. The β-galactosidase expressed from the LacZ gene cleaves the S-gal in the media to leave a black precipitate. The mutagenized residue in Cph8, T541, influences the phosphotransfer reaction mediated by Cph8.

Fig 2. Bacterial photographs prepared with wild type and mutant strains reveal changes in visual contrast.

Bacterial photographs were prepared in solid media for the strains indicated in the upper left corner of each panel using an identical image printed on transparency films. Incubation conditions consisted of red filtered light (0.08–0.15 W/m², 650 nm range) at 37°C for 24 hours. WT: wild type, KD: kinase dead (H534A).

Fig 3. Comparison of light and dark β-galactosidase activity between wild type, kinase dead and mutant strains.

Miller assays were performed in duplicate to measure β-galactosidase activity in Miller Units, as described in the Materials and Methods. The difference in β-galactosidase activity in the dark versus light (fold change) was calculated by dividing activity in the dark by activity in the light of the wild-type, kinase dead and mutant strains. Standard deviations are shown.

Fig 4. Amino acid sequences of mutants selected from Cph8 mutagenized screen.

DNA sequencing was performed on DNA from the wild type strain (WT), the kinase dead strain (H537A), and five colonies selected from the Cph8 mutant hunt. The residue targeted by site-directed mutagenesis, T541, is highlighted in blue, and the H537A mutation expressed by the kinase dead strain is highlighted in green. An additional point mutation found at position 528 in strain T541L is highlighted in red. DNA sequences were not compared outside of the region encoding residues 524–545.

Fig 5. Western blot and densitometric analysis of Cph8 levels in T541S strain.

Samples of the T541S strain, wild type strain, kinase dead strain and NB462, a strain lacking pCph8, were grown in light (odd numbered lanes) or dark (even numbered lanes) and compared with Western analysis. The expected band size of Cph8 is 83 kDa, indicated with a black arrow. (A) Lanes 1–2 are NB462, serving as a negative control for Cph8 protein. Lanes 3–4 contains wild-type Cph8, lanes 5–6 contains kinase dead and lane 7–8 is T541S mutant. Lane 9 (L) contains a Kaleidoscope ladder (Bio-Rad, Hercules, CA, USA). Lane 10 (+) is a truncated EnvZ protein tagged with His6, which serves as a positive control. (B) ImageJ densitometric analysis was used to calculate the relative darkness of sample bands through normalization to the optical intensity of the WT strains. This analysis resulted in quantitative approximations of the relative amounts of Cph8 protein produced per cell for each strain, as shown in the table.