Coupling between the basic replicon and the Kis-Kid maintenance system of plasmid R1: modulation by Kis antitoxin levels and involvement in control of plasmid replication

Abstract

kis-kid, the auxiliary maintenance system of plasmid R1 and copB, the auxiliary copy number control gene of this plasmid, contribute to increase plasmid replication efficiency in cells with lower than average copy number. It is thought that Kis antitoxin levels decrease in these cells and that this acts as the switch that activates the Kid toxin; activated Kid toxin reduces copB-mRNA levels and this increases RepA levels that increases plasmid copy number. In support of this model we now report that: (i) the Kis antitoxin levels do decrease in cells containing a mini-R1 plasmid carrying a repA mutation that reduces plasmid copy number; (ii) kid-dependent replication rescue is abolished in cells in which the Kis antitoxin levels or the CopB levels are increased. Unexpectedly we found that this coordination significantly increases both the copy number of the repA mutant and of the wt mini-R1 plasmid. This indicates that the coordination between plasmid replication functions and kis-kid system contributes significantly to control plasmid R1 replication.

Figure 1

Localization of maintenance regions in the mini-R1 plasmid pKN1562. Horizontal and vertical projections correspond respectively to the basic replicon module and the kis-kid antitoxin-toxin system. The relative positions of key genes in these modules and the promoters involved in RNA synthesis are also indicated.

Figure 2

Inmunodetection of Kis antitoxin levels. Western blot (A,C) and densitometric analysis (B,D) of Kis levels determined in C600 or in its clpP⁻ derivative (C600−∆clpP). Cells contained either the pKN1562 mini-R1 plasmid coding for RepAwt or its repA55 mutant. The repA55 mutation changes R for H in codon 55 of RepA and this results in a thermosensitive replication protein that, at the permissive temperature, reduces plasmid copy number. An equal amount of total protein extracts were loaded in each lane of the gels and the inmuno-signal of the 3×FLAG labeled Kis was densitometred. Values in B and D represent the average of seven independent densitometries and are corrected for the number of plasmid-containing cells. This percentage was 100% for cells containing the wt plasmid and 78% for cells containing the repA55 plasmid replication mutant. * or *** indicate differences which p-values are = 0.01–0.05 or <0.001 respectively.

Figure 3

Effects of stabilizing Kis antitoxin (A) on plasmid replication and (B) on kis-kid transcriptional levels. Analyses were done in a wt strain or in an isogenic clpP⁻ background using plasmids KN1562 and its repA55 thermo-sensitive replication mutant; kid + and kid − indicates respectively analysis carried out in the presence of a pKN1562 or of its kid75 mutant pJLV01. (A) Plasmid copy number determinations (PCN) of pKN1562 (kid +) and its kid75 mutant (kid −) in the presence/absence of a functional clpP gene (clp + or clp −). Values were corrected by the number of plasmid free cells and referred to PCN of the lpp chromosomal gene (see Materials and Methods). The PCN value corresponding to pKN1562 in the wt strain (clp +, kid +) was given the reference value of 1; (B) Transcriptional levels of kis-kid per plasmid copy corresponding to the samples analyzed in panel (A). Transcriptional levels of the lpp gene were used as the reference. *** indicates differences which p-values are <0.001.

Figure 4

Effects of overproducing Kis antitoxin on the modular coupling. pLNBAD-kis is a conditional overproducer of the Kis antitoxin and + or − indicates respectively presence or absence of inducer (arabinose). As a control, analyses were carried also in the presence of the empty vector (pLNBAD). Effects on KN1562 wt, its repA55 mutant and on derivatives of these plasmids carrying the kid17 mutation were determined. (A) Copy number determinations. The PCN value corresponding to pKN1562 was given the reference value of 1; (B) kis-kid transcriptional levels. Values were corrected by the number of plasmid free cells and referred to the PCN or the transcriptional level of the lpp chromosomal gene (see Materials and Methods). * or *** indicate differences which p-values are = 0.01–0.05 or <0.001 respectively.

Figure 5

Effects of abolishing the coupling between maintenance modules on plasmid stability. Plasmid stability was followed determining the percentage of cells retaining the kanamycin resistance marker of pKN1562 and its derivatives after propagation at 30 °C for 30, 60 and 90 generations in rich non-selective media (see Experimental Section). Panel (A) shows the effect of the clpP⁻ background on plasmid stability and Panel (B) shows the effect of overproducing the Kis antitoxin. *** indicate differences which p-values are <0.001.

Figure 6

Effects of an excess of the CopB protein on the efficiency of replication of mini-R1 plasmids. (A) PCN of copB mutants of pKN1562 (kid +) and of its kid75 mutant (kid −) determined in the presence of and overproducer of CopB (pUC18-copB) or of its empty vector (pUC18); (B) Relative PCN values of the wt pKN1562 plasmid and its repA55 mutant. Values were corrected by the number of plasmid free cells and referred to the PCN of the lpp chromosomal gene (see Materials and Methods). Determinations in the presence (+) or absence (−) of CopB overproducer or of its pUC18 vector were done in the presence of a wt kis-kid system (kid +) or of its kid75 mutant (kid −). In (A) and (B), the PCN value corresponding to pKN1562 in the presence of the empty vector (pUC18) was given the reference value of value 1 (A and B). *** indicate differences which p-values are <0.001.