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

Abstract

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.

Polar Positioning of a Conjugation Protein from the Integrative and Conjugative Element ICEBs1ofBacillus subtilis

“…Transfer of the conjugative plasmid R751 in E. coli can occur along any orientation between donors and recipients that are in direct contact, suggesting that the conjugative machinery of R751 may assemble along both the lateral and polar sides of the cell (36). This type of lateral and polar localization of the mating machinery has been directly observed for the R27 conjugative plasmid in E. coli (22,25). R27's VirB4-like TrhC and coupling protein TraG were distributed at multiple sites along all sides of the cell.…”

Conjugal Transfer of a Virulence Plasmid in the Opportunistic Intracellular Actinomycete Rhodococcus equi

“…The effect of incubation temperature on transfer frequency was also reported in Mycobacterium smegmatis and Salmonella enterica serovar Typhi (19,41). In studies of conjugation in M. smegmatis, temperatures ranging from 25°C to 40°C were evaluated, and 30°C was found to be optimal for transfer (41).…”

“…In studies of conjugation in M. smegmatis, temperatures ranging from 25°C to 40°C were evaluated, and 30°C was found to be optimal for transfer (41). Similarly in S. enterica serovar Typhi, 27°C was more conducive for plasmid transfer than 37°C (19). The lower temperatures perhaps better mimic the natural environmental temperatures (e.g., soil and water) at which these organisms generally exist and where conjugation most often occurs.…”

Biological Diversity of Prokaryotic Type IV Secretion Systems

“…An integral membrane association might not be obligatory, however, because VirB4-like TrwK R388 cofractionates with the membrane and the cytosol, and soluble TrwK R388 exhibits ATPase activity in vitro (9). Furthermore, TraC F and TrhC R27 associate with the inner membrane but probably peripherally through interactions with other T4SS subunits (109,234).…”