SENTINEL PLANTS UTILIZING BACTERIAL TWO-COMPONENT SIGNALING SYSTEMS

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: TERMINATED
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0193478
• Project No.: FLA-MCS-04073
• Project Start Date: Jul 1, 2002
• Project End Date: Jun 30, 2005

Project Director
Ingram, L. O.

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
MICROBIOLOGY & CELL SCIENCE

Non Technical Summary
Bacteria and plants offer many advantages as remote (covert or overt) sensors of chemicals in the environment. Native recognition systems can be modified to detect analytes and solutes of specific interest such as TNT/DNT, a component of many explosives. This project explores the feasibility of mutating bacterial two-component systems for use in plants to generate discrete signals to detect novel analytes and solutes.

Animal Health Component

(N/A)

Research Effort Categories

Basic

80%

Applied

(N/A)

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2499	1030	10%
201	2499	1040	15%
201	4010	1000	10%
201	4010	1040	15%
723	2499	1030	10%
723	2499	1040	15%
723	4010	1000	10%
723	4010	1040	15%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
2499 - Plant research, general; 4010 - Bacteria;

Field Of Science
1030 - Cellular biology; 1040 - Molecular biology; 1000 - Biochemistry and biophysics;

Keywords

signal transduction

agrobacterium

arabidopsis

indicator plants

detection

sensors

plant physiology

plant genetics

human health

molecular biology

bacterial physiology

indicator organisms

phosphates

protein kinase

receptors

evolution

protein characterization

protein structure

environmental pollution hazard

promoters (genetics)

protein dna interactions

bacterial genetics

mutagenesis

Goals / Objectives
The goal of this project is to develop a series of interchangeable bio-modules for the detection of specific analytes and solutes via cell based circuits that generate discrete signals. Bacterial systems will be established for mutation and selection of two-component signal transduction systems that recognize TNT/DNT. Systems possessing the desired analyte recognition properties will be transferred and adapted for function in plants (Arabidopsis). These bio-modules will comprise the following: 1) Analyte Sensing Module: the N-terminal portion of the receptor kinase that directly interacts with the analyte. 2) Signal Transduction Module: comprised of domains from two proteins, the transmitter domain of the receptor kinase and the response regulator protein (receiver and DNA binding domains). This module transfers the signal from the receptor kinase to the promoter of the reporter gene via an activated response regulator. 3) Signal Reporter Module: comprised of an inducible promoter regulated by the two-component system and a transcribed region that can encode a variety of reporter genes including enzymes that form colored products, luminescent proteins, or resolvase systems that rearrange DNA.

Project Methods
I. Analyte Sensor Modules- A bacterial two-component system to recognize TNT/DNT will be developed from mutated native two-component systems that recognize analytes with similar chemical structure. The receptor kinase genes will be mutated, isolated and transformed into a stable host where desired clones will be identified by their response to a cocktail of analytes by induction of b-galactosidase. Future screening will identify clones responsive to individual compounds. Bacterial mutagenesis and directed evolution approaches utilizing mutagenic host strains, error-prone PCR and DNA shuffling will be employed to screen and select for optimum analyte recognition and induction properties of the two-component systems. II. Plant Signal Transduction and Reporter Modules-- Initial focus will be on the modification of the A. tumefaciens VirA-VirG-ChvE system to transduce the analyte-binding signal of the receptor kinase (VirA) to the eukaryotic transcription apparatus. The response regulator (VirG) will be modified to become a strong transcriptional activator for plants by creating a VirG-Vp16 fusion protein. The VirA-VirG pathway may be adapted later to function in combination with other two-component systems. Alternatively, each receptor kinase-response regulator pair will be individually adapted for plant function. An important issue will be the correct insertion of the receptor kinase into the plasma membrane of the plant. The hexose-phosphate (UhpCBA) and the NarX/NarL (nitrate) recognition systems of E. coli will serve as alternative two-component systems that will be developed to maximize possibilities for TNT/DNT recognition. Parallel studies conducted on these three systems should provide many opportunities for domain swapping and creation of novel hybrids. Once constructed, these hybrid systems will be passed through repeated cycles of mutagenesis and selection/screening in the development of novel sensing capabilities and signaling pathways. III. Plant Reporters-- Reporter systems will initially be engineered to drive GUS expression in plants but can be readily modified to express a variety of alternative genes to report sensor binding as measured by enzymatic activity, leaf senescence or pigmentation changes, etc. Modifications of the reporter module will be primarily limited to the control of transcription and relatively independent of the gene(s) being expressed.

Progress 07/01/02 to 06/30/05

Outputs
CONCLUSIONS: 1) Although the VirA/VirG systems can be made to become more responsive to TNT, induction was only 2-5 fold, which is below the level of induction and sensitivity required to be useful as part of a biosensor for the presence of TNT in the soil. Much more effort will be needed to obtain mutants of highly sensitive and specific recognition of TNT by mutated VirA protein. 2) Screening for Agrobacteria VirA/VirG mutants in E. coli is useful in terms of increasing transformation efficiencies; however, the VirG-RNA polymerase interactions pose a problem in gene activation. This issue can be partially alleviated by transferring the Agrobacterium alpha subunit of RNA polymerase to E. coli. 3) The bacterial VirG protein can function in plants after addition of a eukaryotic transcriptional activation motif (VP16 in our case) when used in conjunction with an engineered Vir promoter element located upstream of a minimal plant promoter (CaMV 35S TATAA). 4) Modifying the Agrobacterium VirA receptor kinase to function in plants will take more effort. The length of transmembrane regions of receptor proteins seems to differ between bacteria and eukaryotes. This difference is thought to reflect the differences in membrane thickness between these two groups of widely divergent organisms. These differences in the membranes of bacteria and plants caused unanticipated difficulties in demonstrating function for VirA in Arabidopsis plants. Another problem was the insolubility of the VirA protein when expressed in plant cells. Our results indicated that protein bodies, similar to inclusion bodies in bacteria, formed when VirA was expressed in a transient assay system employing onion epithelial cells. The combination of poor overall solubility of the protein in plant cells and the requirement for a major engineering effort directed towards transmembrane domains proved too great an obstacle in our attempts to obtain VirA function in Arabidopsis.

Impacts
The significance of our results is best viewed as a survey of the potential for the Agrobacterium VirA/VirG to be adapted to function in plants as a biosensor system. It appears that the most difficult step will be the adaptation of the VirA and VirG proteins to function in the plant cell in sensing and signal transfer roles, since this requires that the VirA and VirG proteins be present in a functional complex that is partially embedded in the eukaryotic plasmalemma.

Publications

• Jung, C.-Y., Gu, Y., Wu, D. and Jin, S. (2004) Mutants of Agrobacterium tumefaciens virG gene that activate transcription of vir promoter in Escherichia coli, Cur. Microbiol. 49:334-340.

Progress 10/01/01 to 10/01/02

Outputs
This purpose of this work is to develop bacteria and plants which will provide measurable responses to the presence of explosives in soil. Initial work has begun to develop genetic systems which can be used to control these responses and to identify appropriate reporters.

Impacts
Success of this work could greatly facilitate the remediation of contaminated land by guiding efforts to regions with highest concentrations. Other potential applications include serving as remote sensors and reports. Proof of concept with explosives will pave the way for the sensing of many environmentally important chemicals.

Publications

• No publications reported this period