Cellular location and temperature-dependent assembly of IncHI1 plasmid R27-encoded TrhC-associated conjugative transfer protein complexes

Conjugal transfer of IncHI plasmid DNA between Gram-negative bacteria is te mperature sensitive, as mating is optimal between 22 degreesC and 30 degree sC but is inhibited at 37 degreesC. R27, isolated from Salmonella enterica serovar Typhi, is an IncHI1 plasmid of 180kbp that has been sequenced compl etely. 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 micr oscopy allowed visualization of the TrhC-GFP fusion protein, and Escherichi a coli cells were examined for the subcellular localization and temperature -dependent production of TrhC-GFP. At 27 degreesC, TrhC-GFP was found at th e periphery of cells as discrete foci, indicating an association of TrhC wi thin protein complexes in the bacterial cell membrane, whereas at 37 degree sC, little fluorescence was detected. These foci probably represent the int racellular position of protein complexes involved in conjugative transfer, as the formation of foci was dependent upon the presence of other Mpf prote ins. During temperature shift experiments from 37 degreesC to 27 degreesC, a long lag period was required for generation of GFP foci. Conversely, duri ng short shifts from 27 degreesC to 37 degreesC, the GFP foci remained stab le. These results suggest that the expression of transfer genes in the Tra2 region of R27 is temperature dependent. Subcellular localization of TrhC w as verified by cellular fractionation. Expression patterns of TrhC-GFP were confirmed with immunoblot analysis and reverse transcriptase-polymerase ch ain reaction (RT-PCR). These results allow us to propose mechanisms to expl ain the temperature-sensitive transfer of R27.