The rifampicin‐inducible genes srn6 from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by kiiling of plasmid‐free segregants

Abstract

The gene systems srnB of plasmid F and pnd of plasmid R483 were discovered because of their induction by rifampicin. Induction caused membrane damage, RNase I influx, degradation of stable RNA and, consequently, cell killing. We show here that the srnB and pnd systems mediate efficient stabilization of a mini-R1 test-plasmid. We also show that the killer genes srnB' and pndA are regulated by antisense RNAs, and that the srnC- and pndB-encoded antisense RNAs, denoted SrnC- and PndB-RNAs, are unstable molecules of approximately 60 nucleotides. The srnB and pndA mRNAs were found to be very stable. The differential decay rates of the inhibitory antisense RNAs and the killer-gene-encoding mRNAs explain the induction of these gene systems by rifampicin. Furthermore, the observed plasmid-stabilization phenotype associated with the srnB and pnd systems is a consequence of this differential RNA decay: the newborn plasmid-free cells inherit the stable mRNAs, which, after decay of the unstable antisense RNAs, are translated into killer proteins, thus leading to selective killing of the plasmid-free segregants. Thus our observations lead us to conclude that the F srnB and R483 pnd systems are phenotypically indistinguishable from the R1 hok/sok system, despite a 50% dissimilarity at the level of DNA sequence.

Small Toxic Proteins and the Antisense RNAs That Repress Them

“…The main characteristics of Hok-Sok regulation, i.e., RNA processing and the presence of an overlapping open reading frame, appear to be true for all of the other type I toxinantitoxin loci carried on plasmids found in the gram-negative bacteria (36). Gerdes and Wagner also have suggested that synthesis of the chromosomally encoded Ldr toxins is regulated in a manner similar to that for Hok (23)…”

“…Two other loci encoding HokSok-like systems, SrnB-SrnC (stable RNA negative) of the F plasmid (37,39) and PndA-PndB (promotion of nucleic acid degradation) of plasmid R483 (2,38), were noted when it was observed that the addition of rifampin led to membrane damage, RNase I influx from the periplasm, degradation of stable RNA, and cell killing (29). It was subsequently shown that the arrangement of genes was very similar to that for Hok-Sok (20), with srnB and pndA encoding toxin proteins containing single transmembrane domains and srnC and pndB encoding small antisense RNAs (36). RNAI-RNAII encoded on the pAD1 plasmid of Enterococcus faecalis, the first type I toxinantitoxin pair found for a gram-positive bacterium, was identified on the basis of a postsegregational killing phenotype (54,55).…”

Conditionally lethal genes associated with bacterial plasmids

“…These loci were originally identified from their ability to induce membrane damage, RNase I influx, degradation of stable RNA and eventually cell killing upon addition of rifampicin. It was shown later by Nielsen et al (1991) that these genes in fact mediate post-segregational killing of plasmid-free cells, are regulated by antisense RNAs, and that they also share the same structural and functional organization as hoklsok (Fig. 3).…”

Hypothesis: type I toxin–antitoxin genes enter the persistence field—a feedback mechanism explaining membrane homoeostasis

“…The answer to this question was unravelled in a long series of papers in which we focused on the hok-sok interaction. The first clue to the mechanism came from the observation that hok mRNA exists in two forms: an inactive full-length, primary transcript of 400 nucleotides (nt) and a 3 0 -end processed, translationally active mRNA of 360 nt [54,62]. Using both genetic, phylogenetic and biochemical approaches, we identified several regulatory elements in hok mRNA: we showed that the 5 0 -end of full-length hok mRNA base-pairs with the very 3 0 -end and that the resulting, highly folded mRNA is inactive with respect to both Sok-RNA and ribosome binding.…”