Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity

Abstract

Although the broad-host-range IncP plasmids can vegetatively replicate in diverse gram-negative bacteria, the development of shuttle vector systems has established that the host range for IncP plasmid conjugative transfer is greater than the range of bacteria that sustain IncP replicons. Towards understanding IncP plasmid conjugation and the connection between IncP conjugation and Agrobacterium tumefaciens T-DNA transfer to plants, two sets of mutants were generated in the larger transfer region (Tral) of the IncPa plasmid RK2. Mutagenesis strategies were chosen to minimize transcriptional polar effects. Mutant Tral clones were mapped, sequenced, and processed to reconstruct 49.5-kb Tra2-containing plasmid derivatives in order to assay for transfer activity and IncP plasmid-specific phage sensitivity. Focusing on the activities of the gene products of traF and traG in Escherichia coli, we found that mutations in traF abolished transfer activity and rendered the host cells phage resistant and mutations in traG abolished transfer activity but had no effect on phage sensitivity. Complementation of these mutant derivatives with corresponding trans-acting clones carrying traF or traG restored transfer activity and, in the case of the traF mutant, the phage sensitivity of the host cell. We conclude that in E. coli, both TraF and TraG are essential for IncP plasmid transfer and that TraF is necessary (but not sufficient) for donor-specific phage sensitivity, and sequencing data suggest that both TraF and TraG are membrane spanning.The remarkable broad-host-range transfer properties of the IncP plasmids have stimulated investigation of the basic mechanism of conjugation as well as the use of these plasmids as tools for genetic manipulation of a wide variety of bacteria. The IncP plasmids can replicate in diverse gram-negative organisms, but the host range defined by conjugative transfer ability is considerably greater than the range of vegetative replication proficiency (15,19,50). With the construction of shuttle vector systems consisting of more than one host-specific replication system and the IncP transfer system, plasmid derivatives have been shown to transfer from gram-negative bacteria to highly divergent gram-negative organisms (17, 30, 52), to acid-fast bacteria (26), to gram-positive bacteria (47), and to yeast (22). In addition, significant similarities have recently been described between the IncP conjugation system and the T-DNA and vir loci of Agrobacterium tumefaciens tumor-inducing (Ti) plasmids that promote DNA transfer to plants (49, 56). These similarities are underscored by the recent demonstration that theA. tumefaciens Vir region can mobilize the small non-selftransmissible IncQ plasmids (2) among agrobacteria in a manner similar to the established IncP mobilization of these IncQ plasmids (11).Unlike the F plasmid, in which the conjugative transfer genes are organized into a single contiguous operon (51), the IncP plasmids have a more complicated arrangement of their transfer systems. In the IncPa ...

The mating pair formation system of plasmid RP4 defined by RSF1010 mobilization and donor-specific phage propagation

“…Both Tral and Tra2 were essential to mobilize the IncQ plasmid RSF1010 and for propagation of donor-specific bacteriophages. Recently, Waters et al (73) identified the traF gene product as one Tral component essential for phage propagation. However, it remained unclear whether additional Tral functions are required for this ability.…”

Inhibition of Agrobacterium tumefaciens oncogenicity by the osa gene of pSa

“…Interestingly, a partial sequence of the traG gene of pKM101 shares similarities to that of the virB1l gene of the Ti plasmid virB operon and is directly upstream of the nuc gene (37). The T-DNA processing genes such as the virD genes also bear sequence homologies to the tra genes of RP4 (28,29,47). Hence, the inhibition of the oncogenicity ofA.…”

Conjugative transfer in the dissemination of beta-lactam and aminoglycoside resistance

“…TraF is required in some specialized way for incoming phage DNA, while TraF and TraG together, possibly in equimolar amounts, are required for the exiting of conjugating DNA from the donor cell (table 4). Complementation of mutant RK2 donors (traF or traG) can be accomplished by the corresponding gene in the donor and not in the recipient (33,49).The concept of membrane fusion sites at the transfer bridge, which may also serve as phage adsorption sites, is reminiscent of Bayer's fusion sites. Bayer observed that some T phage appeared to adsorb at such membrane adhesion sites, visualized by electron microscopy as points of contact between the inner and outer membranes of plasmolyzed E. coli cells (56).…”

“…The connecting link between DNA preparation and the mating bridge may be supplied by the two RK2 proteins TraF and TraG, and their analogs in other systems. In RK2, these two proteins appear to be co-transcribed and co-translated (49,52). The TraG protein has an NTP-binding domain and might thereby provide energy for DNA transport, working together with TraF which has sequences suggesting a role as a DNA processivity factor (53,54).…”

“…The TraG protein has an NTP-binding domain and might thereby provide energy for DNA transport, working together with TraF which has sequences suggesting a role as a DNA processivity factor (53,54). Both TraF and TraG are absolutely essential for conjugation (49). The relaxosome may begin transfer by interacting with TraF and TraG, which are either closely associated with, or are part of, the mating bridge.…”