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Fig. 2 | BMC Microbiology

Fig. 2

From: The role of the two-component systems Cpx and Arc in protein alterations upon gentamicin treatment in Escherichia coli

Fig. 2

CpxA and ArcA interact in vivo. a Bacterial two-hybrid assay (BACTH) was used to identify interactions between CpxA and ArcA. The sensor kinases CpxA and ArcB were C-terminally fused to the T18-fragment, whereas the response regulators CpxR and ArcA were N-terminally fused to the T25-fragment of the Bordetella pertussis adenylate cyclase. Different combinations of sensor kinase and response regulator were co-overexpressed and the ß-galactosidase activity in Miller units [MU] was determined. CpxA/CpxR- and ArcB/ArcA-interactions serve as controls. Plasmids expressing fusions of T18 and T25 with leucine zipper domains of the transcription factor GCN4 derived from Saccharomyces cerevisiae (+) serve as a positive control. Empty vectors non-expressing the T18- and T25-fragment (−) serve as a negative control. The average of three biological replicates is shown. b The chromosomally-encoded fusion protein ArcA-Snap and plasmid-encoded CpxA-Strep were used to analyze interactions between CpxA and ArcA using Membrane-SPINE. Interactions were determined for cells treated with or without of 5 μg ml−1 gentamicin for 40 min. Additionally, samples without addition of the crosslinker formaldehyde or gentamicin served as controls. Out of two biological replicates, one representative blot is shown. Black triangles show specific bands of CpxA-Strep and ArcA-Snap, whereas white triangles represent unspecific bands

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