PifC and Osa, Plasmid Weapons against Rival Conjugative Coupling Proteins

Abstract

Bacteria display a variety of mechanisms to control plasmid conjugation. Among them, fertility inhibition (FI) systems prevent conjugation of co-resident plasmids within donor cells. Analysis of the mechanisms of inhibition between conjugative plasmids could provide new alternatives to fight antibiotic resistance dissemination. In this work, inhibition of conjugation of broad host range IncW plasmids was analyzed in the presence of a set of co-resident plasmids. Strong FI systems against plasmid R388 conjugation were found in IncF/MOB_F12 as well as in IncI/MOB_P12 plasmids, represented by plasmids F and R64, respectively. In both cases, the responsible gene was pifC, known also to be involved in FI of IncP plasmids and Agrobacterium T-DNA transfer to plant cells. It was also discovered that the R388 gene osa, which affects T-DNA transfer, also prevented conjugation of IncP-1/MOB_P11 plasmids represented by plasmids RP4 and R751. Conjugation experiments of different mobilizable plasmids, helped by either FI-susceptible or FI-resistant transfer systems, demonstrated that the conjugative component affected by both PifC and Osa was the type IV conjugative coupling protein. In addition, in silico analysis of FI proteins suggests that they represent recent acquisitions of conjugative plasmids, i.e., are not shared by members of the same plasmid species. This implies that FI are rapidly-moving accessory genes, possibly acting on evolutionary fights between plasmids for the colonization of specific hosts.

Keywords: Osa protein; PifC protein; antimicrobial resistance; bacterial conjugation; fertility inhibition; mobilization; plasmid; type IV coupling protein.

Figure 1

Effect of pifC on plasmid conjugative transfer. The conjugation frequencies of plasmids R388, pMBUI4, pRL443, R751, pKM101, and pOX38 in the presence (blue squares) or absence (yellow circles) of pifC in donor cells are shown. Each point represents the conjugation frequency (T/D) obtained in one independent experiment. Horizontal and vertical bars represent the mean ± SD obtained for each group of data (^**p < 0.01, ^***p < 0.001).

Figure 2

Effect of pifC on the transfer of mobilizable plasmids by representative conjugative helpers. The mobilization frequencies of ColE1, RSF1010, and CloDF13, using either R388, R751, pRL443, or pKM101 as helpers, in the presence (blue squares) or absence (yellow circles) of pifC in donor cells, are shown. Each point represents the mobilization frequency (T/D) of one independent experiment. Horizontal and vertical bars represent the mean ± SD obtained for each group of data (^***p < 0.001).

Figure 3

Modulation of the PifC-mediated FI of R388 by overexpression of the coupling protein. Each point represents the conjugation frequency (T/D) of one independent experiment, in the presence or absence of pOX38 and R388 coupling protein TrwB. Horizontal and vertical bars represent the mean ± SD obtained for each group of data (^***p < 0.001). None/- (indicated by yellow circles), R388 alone. None/pBAD33::trwB (yellow circles), R388 in the presence of pBAD33::trwB. pOX38/- (blue squares), R388 in the presence of pOX38::KmR. pOX38/pBAD33 (blue squares), R388 in the presence of pOX38::KmR and pBAD33 empty vector. pOX38/pBAD33::trwB (orange squares), R388 in the presence of pOX38::KmR and pBAD33::trwB.

Figure 4

Effect of osa on the transfer of conjugative and mobilizable plasmids. (A) FI of IncP-1 plasmids mediated by IncW plasmids. (B) FI of IncP-1 plasmids mediated by osa. (C) Effect of osa on the transfer frequency of mobilizable plasmids by IncP-1 helpers. Each point represents the transfer frequency (T/D) of one independent experiment in the presence (blue squares) or absence (yellow circles) of osa in donor cells. Horizontal and vertical bars represent the mean ± SD obtained for each group of data (^**p < 0.01, ^***p < 0.001).

Figure 5

Abundance of FI protein families in the NCBI plasmid database. The figure shows the number of plasmids that contain one hit to each of the FI protein families. See details in section Materials and Methods and Supplementary Table 2.

Figure 6

Schematic summary of the plasmid interactions observed in this study. Plasmid incompatibility groups are represented by colored circles. Continuous lines show identified fertility inhibition systems from plasmids in white boxes. Dashed lines show fertility inhibition systems caused by unidentified genes from plasmids in white boxes.