Conjugative transfer functions of broad‐host‐range plasmid RK2 are coregulated with vegetative replication

Abstract

The kilB locus (which is unclonable in the absence of korB) of broad-host-range plasmid RK2 (60 kb) lies between the trfA operon (co-ordinates 16.4 to 18.2 kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co-ordinates 20.0 to 27.0 kb). Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter. The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2. We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis. Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids. However insertion in ORFI (kilBI) of the region analysed results in a Tra- phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region. From the kilBI DNA sequence KilBI is predicted to be 34995 Da, in line with M(r) = 36,000 observed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and contains a type I ATP-binding motif. The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium. Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tumefaciens Ti plasmid DNA from bacteria to plant cells. The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI may be involved in assembly of the surface-associated Tra functions. The data presented in this paper provide the first demonstration of coregulation of genes required for vegetative replication and conjugative transfer on a bacterial plasmid.

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

“…The reading frames for traF and traG contain overlapping start/stop codons (23a, 56 (24,40). Functions mapping within Tra2 include phage sensitivity, entry exclusion, and the locus kilB, also designated trbB (28,35). The kilB gene, which has homology with virBIl of A. tumefaciens (35), has been shown by mutation and complementation analysis to be required for transfer (27) (Fig.…”

“…Functions mapping within Tra2 include phage sensitivity, entry exclusion, and the locus kilB, also designated trbB (28,35). The kilB gene, which has homology with virBIl of A. tumefaciens (35), has been shown by mutation and complementation analysis to be required for transfer (27) (Fig. 1) (25, 31) t' appear to adsorb to pili (7,8), it has not been demonstrated that these pili are sex pili or that all phage resistance is due to loss of these pili.…”

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…VirB11 belongs to the family of PulE-like NTPases found in type II, type IV and tad-like secretion systems (Motallebi-Veshareh et al, 1992;Planet et al, 2001;Whitchurch et al, 1991). Along with VirB4 and the CP (VirD4), VirB11 is thought to energize the type IV secretion machinery either for complex assembly, pilus production and/or substrate secretion.…”

Coupling Factors in Macromolecular Type-IV Secretion Machineries

“…The structure of H. pylori Cag525 protein [138], a member of the VirB11/PulE family of ATPases [144] involved in the type-IV secretion system of the latter pathogen, reveals a six-clawed grapple shape mounted on a hexameric ring and it is proposed to be membrane-associated. It has been shown to function as dynamic hexameric assemblies with nucleotide-dependent conformational changes regulating substrate export or T4SS assembly [145].…”