Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila (original) (raw)
Figure 1
Construction of a library expressing fusions of β-lactamase and L. pneumophila hypothetical proteins.
A. A schematic structure of the fusion proteins and the screening strategy. In most cases, the gene was fused with the β-lactamase by inserting into the vector as a _Bam_HI/_Sal_I fragment. After infection, samples were loaded with the CCF4-AM dye and were inspected under a fluorescence microscope. B. Evaluation of the library for expression of the fusion proteins. Plasmids directing expression of β-lactamase fusions were introduced into a wild type L. pneumophila strain. Total cell lysates of bacteria grown in the presence of IPTG were used to examine the steady state of the fusion proteins by immunoblot. In each image, the detection of a protein nonspecifically recognized by the antibody (arrow) was used as a loading control.