Figure 5.

The TolQ amino-terminal domain interacts with the FtsN periplasmic domain. The potential for various regions of the TolQ and FtsN proteins to interact were tested by a two-hybrid analysis, using plasmids encoding the FtsN domains fused to RNαP2 and the TolQ domains fused to lambda cI. The individual regions considered for each protein are depicted by the diagram at the top, and identified by protein and topology relative to the cytoplasmic membrane. Thus, the cytoplasmically exposed FtsN residues 1-33 are termed “FC”, while the periplasmically localized FtsN residues 54-243 are termed “FP”. Similarly, the cytoplasmically exposed TolQ residues 39-135 and 194-230 are termed “TC1” and “TC2”, respectively, while the periplasmically localized TolQ residues 1-19 and 157-174 are termed “TP1” and TP2”, respectively. Indicator cells coexpressing various fusions and/or control products were plated on four different media and scored for growth at 24 h: “A”, LB medium; “B”, nonselective His dropout medium containing chloramphenicol 25 μg mL⁻¹ and tetracycline 12.5 μg mL⁻¹; “C”, selective screening medium comprised of nonselective agar supplemented with 5 mmol/L 3-amino-1,2,4 triazole; and “D”, dual selective screening medium comprised of selective screening agar supplemented with streptomycin 12.5 μg mL⁻¹. Results are displayed as three sets: “Controls”, consisting of indicator cells with no plasmid, a positive control of two fusion proteins known to interact (LGF2/Gall11^P), and a negative control pairing the lambda cI and RNαP2 encoding plasmids lacking fusion domains (cI/RNα); “Cytoplasmic pairings” consisting of indicator cells bearing combinations of plasmids encoding fusions of the cytoplasmic domains of FtsN and TolQ paired with each other or with cognate controls and; “Periplasmic pairings”, consisting of indicator cells bearing combinations of plasmids encoding fusions of the periplasmic domains of FtsN and TolQ paired with each other or with cognate controls.