Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein

Abstract

The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism. A ParE protein derivative, designated ParE, was purified by construction of a fusion protein, GST-ParE, followed by glutathione- In order for a plasmid to be stably maintained in a bacterial population, each daughter cell must inherit at least one copy of the plasmid upon cell division. For high-copy-number plasmids, random segregation alone may be adequate for achieving stability. Low-copy-number plasmids, however, employ one or more genetic loci which encode mechanisms which stabilize the plasmid within a growing bacterial population (49). Several different plasmid stabilization mechanisms have been identified (19,31,56). One such system involves the selective killing of plasmid-free daughter cells of Escherichia coli. The hok/sok, srnB, and pnd loci of plasmids R1, F, and R483, respectively, encode cytotoxic proteins (Hok, SrnBЈ, and Pnd, respectively), which cause a ''ghost'' cell phenotype (14). Expression of the toxin is posttranscriptionally regulated by an antisense RNA, which blocks translation and exhibits a relatively short half-life (13, 15). In a plasmid-free segregant, the unstable antisense RNA is degraded, allowing for translation of the toxins and the subsequent death of the plasmid-free cell (52).In the case of another family of killing systems (23), which includes the ccd locus of F (2,9,20,22,47,51), the parD/pem locus of R1-R100 (4, 5, 39, 54), and the phd-doc system of prophage P1 (28), two proteins are involved, a toxin (CcdB, Kis/PemK, and Doc, respectively) and an antitoxin (CcdA, Kid/PemI, and Phd, respectively), which presumably is more susceptible to degradation in a plasmid-free cell (5, 20, 28). CcdA and PemI protein degradation has been shown to be mediated by the Lon protease (53, 55), while Phd degradation is mediated by the ClpXP serine protease (29). Thus, in the absence of the plasmid, sufficiently high levels of antitoxin are not maintained, and the toxin is able to kill the host cell. Cell death is accompanied by filamentation and has been shown for the ccd system to be caused by the ability of the CcdB protein to induce the ATP-dependent cleavage of DNA by gyrase (3). In the case of the F ccd and R1 parD systems, the toxin and antitoxin are transcribed from the same operon, which is autoregulated by the coordinated action of both proteins (9, 39, 51).RK2 is a broad-host-range plasmid (32) that despite a relatively low copy number of four to seven copies per chromosome (12) is stably maintained in a wide-range of gram-negative bacteria. Stabilization of RK2 is accomplished by at least two genetic regions of the plasmid utilizing different stabilization mechanisms. The psa (postsegregational arrest) locus, which has not been mapped, causes the growth inhibition of daughter cells which fail to inherit the plasmid (24). Factors encoded by this...

Mechanisms of Toxin Inhibition and Transcriptional Repression by Escherichia coli DinJ-YafQ

“…Addition of the YafQ Toxin to the YafQ-(DinJ) 2 -YafQ-DNA Repressor Complex Does Not Alter Its Stability-One proposed additional role of toxin proteins is that upon increasing toxin levels during stress, the toxin functions as a transcriptional derepressor via destabilization of the TA-DNA repressor complex (25)(26)(27)(28). To address whether transcription of the dinJ-yafQ operon is regulated in the same manner, we performed competition EMSAs where we first formed a complex of YafQ- (DinJ) 2 -YafQ bound to PdinJ and then incubated with increasing concentrations of YafQ (Fig.…”

“…Cellular stress leads to an increase in toxin protein accumulation, most likely from antitoxin degradation. Specific toxin proteins such as RelE, Doc, and CcdA have been proposed to function as either transcriptional co-repressors, as part of the TA complex, or as de-repressors, which are dependent upon changes in the toxin-antitoxin ratios (25)(26)(27)(28)(29). Transcriptional de-repression is thought to occur by a mechanism in which excess toxin interacts with and destabilizes the TA-DNA repressor complex (25,26,29).…”

The ParB protein encoded by the RP4 par region is a Ca2+-dependent nuclease linearizing circular DNA substrates

“…The partitioning region (par) comprises two operons, parCBA and parDE, which are transcribed from divergent promoters (Gerlitz et al, 1990;Eberl et al, 1992;Davis et al, 1992). The parDE operon specifies a growth inhibition function mediating plasmid stabilization by cell killing associated with plasmid loss (Roberts et al, 1994;Jensen et al, 1995;Johnson et al, 1996). The parCBA operon appears to encode a complex strated that pBR322 derivatives containing the RP4 par region deleted for both parB and parC but leaving the other functions intact showed reduced stability (Gerlitz et al, 1990).…”

The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins

“…Plasmidcured cells cannot produce the antidote, leaving the stable toxin uncomplexed, which consequently causes cell killing or growth retardation [7]. The parDE operon of broad-host-range plasmid RP4\RK2 constitutes such a plasmid-addiction system and has been characterized in terms of genetics and function [8,9], together with other killing modules of extrachromosomal elements. Recent studies include ccd of plasmid F [10], parD and pem of plasmids R1 and R100 [11], phd\doc of prophage P1 [12] and pas of plasmid pTF-FC2 [4].…”