The Tra2 core of the IncP(alpha) plasmid RP4 is required for intergeneric mating between Escherichia coli and Streptomyces lividans

Abstract

Escherichia coli cells and Streptomyces mycelia are able to form close contacts in the absence of a conjugative system which might facilitate intergeneric plasmid transfer without the genes required for mating pair formation (Tra2) of the RP4 plasmid. The same Tra2 genes found to be essential for RP4 plasmid transfer, RSF1010 mobilization, and donor-specific phage propagation in E. coli were also required for intergeneric transfer between E. coli and Streptomyces lividans.Bacterial conjugation, the horizontal transmission of genetic material, usually requires close contact between donor and recipient cells, as demonstrated for F-plasmid-mediated transfer (4). However, it has been suggested that F-plasmid transfer can occur through the pilus between donor and recipient cells held at a distance from one another (6, 12). In addition, the demonstration that mating could occur between organisms with very different cell envelopes suggested that the establishment of a productive mating pair need not require tight junction formation.Following reports that RP4-mediated intergeneric plasmid transfer was possible between Escherichia coli and Streptomyces lividans (11), we wished to investigate whether the genes for pilus assembly were required for this event. The RP4 pilus, unlike its F counterpart, is rarely found attached to the cell surface, suggesting that it has no role in initiating contact between cells (5). However, E. coli and S. lividans can form close contacts in the absence of a known conjugation system in either cell (see below). Thus, plasmid transfer might require only the genes for DNA metabolism (Tra1) during conjugation. The requirement of genes for pilus formation (Tra2) would be evidence supporting an important role for the pilus in DNA transfer.Adhesion between E. coli and S. lividans. Examination of mixtures of E. coli and S. lividans with a scanning electron microscope revealed that E. coli was closely associated with the mycelial surface, usually via the pole of the E. coli cell. This phenomenon was observed with E. coli S17-1 (14) and S. lividans 1326 (7) (Fig. 1A) as well as other strains of E. coli and S. lividans (data not shown). The presence of a conjugative system in either strain did not have an effect on the ability of the cells to adhere to one another. Further examination by transmission electron microscopy of samples prepared by cryofixation (4) revealed a defined structure between the two cells (Fig. 1B) which resembled the F-plasmid-mediated conjugative junctions visualized in thin sections of mating E. coli cells (4). We were intrigued by these close contacts and wondered if they could substitute for the functions that encode mating pair formation in RP4 during intergeneric plasmid transfer from E. coli to S. lividans.Mobilization of pSET152 from E. coli to S. lividans. Intergeneric transfer of pSET152 (contains the multiple cloning site and replicon of pUC plasmids, the origin of transfer [oriT] of RK2, int C31 of the Streptomyces phage C31, and the apramycin resistance marker which ...

Conjugative Junctions in RP4-Mediated Mating of Escherichia coli

“…While RP4 transfer genes have been characterized (9,11,13,16,19), studies of the structural association between bacterial cells in RP4-mediated matings have been lacking. To use conventional microscopy techniques to study solid-substrate matings is difficult, as traditional chemical fixation methods have employed immersion in liquid, which could disrupt cell-cell associations.…”

“…The donor strains were E. coli HB101 containing either no plasmid (nonmating control), RP4, or various derivatives of RP4 (Table 1); the recipient strains were E. coli SCS1 (Rif r ). These strains are morphologically indistinguishable, a disadvantage, but these matings exactly reproduced the conditions used in earlier biochemical, molecular, and physiological studies (4,7,9,11,13,14,19).To prepare samples for electron microscopy, the protocol used for quantitative filter matings was followed (19). Overnight cultures from frozen stocks were grown in YT medium containing the appropriate antibiotics for the plasmid(s) carried by each strain (Table 1).…”

Incompatibility Protein IncC and Global Regulator KorB Interact in Active Partition of Promiscuous Plasmid RK2

“…They are capable of promoting conjugative transfer to diverse organisms, including gram-negative and gram-positive bacteria and even some yeast species (14,31,35,36,67,87). In addition, IncP plasmids are maintained as stable, autonomously replicating elements in a wide variety of gram-negative hosts (79,83).…”

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp . Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

“…In all these cases, the donor species was E. coli. The conjugative functions of plasmid RP4, (5,10,16,18,21,26,32), the RP1 derivative pMB307 (8), and the RK2 derivative pUZ8002 (23) were used to mobilize the resident plasmids. Several cloning vectors which could be transferred from E. coli to Streptomyces spp.…”