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Conjugal transfer of IncHI plasmid DNA between Gram-negative bacteria is temperature sensitive, as mating is optimal between 22°C and 30°C but is inhibited at 37°C. R27, isolated from Salmonella enterica serovar Typhi, is an IncHI1 plasmid of 180 kbp that has been sequenced completely. The gene encoding green fluorescent protein (GFP) was inserted into R27 in frame with trhC. TrhC is a mating pair formation (Mpf) protein that is essential for plasmid transfer and H-pilus production. Fluorescence microscopy allowed visualization of the TrhC–GFP fusion protein, and Escherichia coli cells were examined for the subcellular localization and temperature-dependent production of TrhC–GFP. At 27°C, TrhC–GFP was found at the periphery of cells as discrete foci, indicating an association of TrhC within protein complexes in the bacterial cell membrane, whereas at 37°C, little fluorescence was detected. These foci probably represent the intracellular position of protein complexes involved in conjugative transfer, as the formation of foci was dependent upon the presence of other Mpf proteins. During temperature shift experiments from 37°C to 27°C, a long lag period was required for generation of GFP foci. Conversely, during short shifts from 27°C to 37°C, the GFP foci remained stable. These results suggest that the expression of transfer genes in the Tra2 region of R27 is temperature dependent. Subcellular localization of TrhC was verified by cellular fractionation. Expression patterns of TrhC–GFP were confirmed with immunoblot analysis and reverse transcriptase–polymerase chain reaction (RT–PCR). These results allow us to propose mechanisms to explain the temperature-sensitive transfer of R27.