Overview

Escherichia coli is both an important pathogen and a primary model organism for microbial biology. Nevertheless, about half of its proteins have not been characterized at any significant level of detail. We are performing an exhaustive yeast two-hybrid screen to comprehensively describe all protein-protein interactions in E. coli. This research will add significantly to recent attempts to characterize purified protein complexes by mass spectrometry (MS) and other methods. By contrast to the MS studies we focus on direct binary interactions.

We have cloned all E.coli open reading frames (ORFs) into Gateway entry clones as well as two-hybrid bait and prey vectors and will test them for all pairwise combinations in an automated yeast two-hybrid system. In addition to testing all full-length proteins, selected protein domains will be searched for interactions. This will allow us to map binding sites to the amino acid level and subsequently study their atomic details using existing crystal or NMR structures.

As part of this project we have already developed new two-hybrid vectors and protocols. This was necessary as the yeast two-hybrid system typically detects only a fraction of all interactions.

The goal of the project is to deliver a comprehensive molecular description of all protein-protein interactions in E. coli. Such maps can be used to integrate other data about metabolic reactions, gene regulation, and signaling networks to achieve a system level understanding of a bacterial cell. Eventually this information will allow us predict the behavior of a cell by computer modeling. With such a level of understanding, we will be both able to target defined processes therapeutically by antibiotics as well as modify the metabolism of the cell in order to use bacteria as chemical factories.

Publications

Funding

NIH / National Institute of General Medical Sciences

Rajagopala Venkatappa