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

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