AHK4 Assay

Introduction

To establish a co-culture system, it is important that E. coli can respond to signals produced by human cells. In our project, we decided to use isopentenyl adenine (iP), a cytokinin, as the signals and AHK4, a receptor of cytokinins exists in Arabidopsis thaliana, as the receptor. This AHK4 can respond to iP by using a histidine-to-aspartate phosphorelay system existing in E. coli.

A histidine-to-aspartate phosphorelay system is one of the most important signal transduction systems for prokaryotes to respond to environmental stimuli. This system includes two important components: a histidine kinase and a response regulator. The histidine kinase has a sensor domain which receives an environmental stimulus. After the histidine kinase sense a stimulus, it autophosphorylates and then the phosphate group is transferred to the response regulator, which in turn, promote expression of a certain gene corresponding to the stimulus.

One of the His-to-Asp phosphorelay systems used in E. coli is composed of three components: RcsC, a histidine kinase, RcsD, a histidine-containing phosphotransmitter, and RcsB, a response regulator. In this system, cps operon is activated through the pathway of RcsC→RcsD→RscB→cps. Previous study (Suzuki et al. 2001) showed that AHK4, a histidine kinase, can also take advantage of RcsD→RscB→cps pathway in E. coli by receiving cytokinins.

Since iP and AHK4 are only used in plants, we considered that employing this AHK4→RcsD→RscB→cps pathway enable us to establish communication between human cells and bacteria without activating any other unexpected genes.

Summary

The purpose of experiments on this page is to confirm that AHK4 can receive iP, a signal molecule produced by human cells, and AHK4→RcsD→RscB→cps pathyway will be activated in turn. To see the activation of the pathway, we used KMI002 strain as a carrier of AHK4. This KMI002 possesses cps::lacZ fusion gene and the activation of AHK4→RcsD→RscB→cps::lacZ pathway can be observed through the activity of β-galactosidase.

As a qualitative experiment, we observed it if AKH4 carrying cells develop blue color under the existence of iP and X-gal on agar plates.

As a quantitative experiment, we cultured E. coli with various concentrations of iP in liquid medium and measured β-galactosidase activity by using ONPG.

Results

1. Qualitative experiment

As shown in Fig1, blue color was developed only when cells were carrying AHK4 and when the medium was containing iP. Therefore, we concluded that AHK4 could receive iP and downstream AHK4→RcsD→RscB→cps::lacZ pathway was activated in turn.

Cells were grown at room temperature on LB agar plates with and without iP. β-galactosidase activity was monitored by X-gal. Photographs were taken after 25h incubation.

2. Quantitative experiment

As shown in Fig2, over 1µM of iP is required to see a difference of β-galactosidase activity between AHK4 carrying cells and negative control cells. The β-galactosidase activity induced by 100µM iP was 2.03-fold higher than the activity induced by 1µM iP.

Cells were grown in liquid LB medium containing various concentrations of iP for overnight at 25℃ at 900 rpm. β-galactosidase activity was monitored by ONPG.

3. Others

In our assay, we used bad/araC promotor, a L-Arabinose inducible promotor, for the expression of AHK4. Therefore, we fist tried to determine appropriate L-Arabinose concentration. But through the experiment, we found following two big problems caused by adding L-Arabinose in medium.

1. cps promoter was induced by different pathway from AHK4→RcsD→RscB→cps::lacZ pathway under the exsistence of L-Arabinose.

2. The growth of AHK4 carrying cells were inhibited by actively expressing AHK4 receptor by L-Arabinose.

Hence, we decided to conduct the experiments without L-Arabinose.

Cells were grown on LB agar plates containing 0.2% L-Arabinose with and without iP at room temperature. Photographs were taken after 25h incubation. Negative control cells developed blue color under the existence of L-Arabinose and the growth of AHK4 carrying cells were inhibited compared with negative control cells.

Discussion

Through the experiments we could confirm that AHK4 can receive iP and cps promoter will be activated in turn. This result showed us that we can control the growth of bacteria by fusing a gene of growth inhibiting factor, such as mazF, downstream the promoter.

However, we need as much as 1µM of iP to see a activity of β-galactosidase as shown in result 2. But other study (Lukáš Spíchal　et al. 2004) showed that 0.1µM of iP can trigger the response of AHK4. Therefore, we consider that we can amplify the output of the pathway by inserting cps promotor and downstream gene into a high-copy plasmid.

For another improvement, we consider that we can slightly increase the expression of AHK4 by using promoter which is leakier than bad/araC promoter, such as lac promoter.

Reference

Suzuki, T., Miwa, K., Ishikawa, K., Yamada, H., Aiba, H. and Mizuno, T. (2001) The Arabidopsis Sensor His-kinase, AHK4, Can Respond to Cytokinins. Plant Cell Physiol. 42: 107-113.

Yamada, H., Suzuki, T., Terada, K., Takei, K., Ishikawa, K., Miwa, K., Yamashino, T. and Mizuno, T. (2001) The Arabidopsis AHK4 Histidine Kinases is a Cytokinin-Binding Receptor that Transduces Cytokinin Signals Across the Membrane. Plant Cell Physiol. 42: 1017-1023.

Spíchal, L., Rakova, N.Y., Riefler, M., Mizuno, T., Romanov, G.A.,Strnad, M. and Schmülling, T. (2004) Two Cytokinin Receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, Differ in their Ligand Specifity in a Bacterial Assay. Plant Cell Physiol. 45: 1299-1305.

Klimeš, P., Turek, D., Mazura, P., Gallová, L., Spíchal, L. and Brzobohatý, B. (2017) High Throughput Screening Method for Identifying Potential Agonists and Antagonists of Arabidopsis thaliana Cytokinin Receptor CRE1/AHK4. Frontiers in Plant Science.

Mizuno, T. and Yamashino, T. (2010) BIOCHEMICAL CHARACTERIZATION OF PLANT HORMONE CYTOKININ-RECEPTOR HISTIDINE KINASES USING MICROORGANISMS. Methods in Enzymology: 335-344.