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

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.     

Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region.

A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization.     

Plasmid genes increase membrane permeability in Escherichia coli

The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids. The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin. These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively. srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product. In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain. Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation. These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.     

Combining the hok/sok, parDE, and pnd postsegregational killer loci to enhance plasmid stability.

To enhance plasmid segregational stability in bacterial cells, two pairs of independent postsegregational killing loci (genes which induce host killing upon plasmid loss) isolated from plasmids R1, R483, or RP4 (hok+/sok+ pnd+ or hok+/sok+ parDE+) were cloned into a common site of the beta-galactosidase expression vector pMJR1750 (ptac::lacZ+) to form a series of plasmids in which the effect of one or two stability loci on segregational plasmid stability could be discerned. Adding two antisense killer loci (hok+/sok+ pnd+) decreased the specific growth rate by 50% though they were more effective at reducing segregational instability than hok+/sok+ alone. With the ptac promoter induced fully (2.0 mM isopropyl-beta-D-thiogalactopyranoside) and no antibiotic selection pressure, the combination of a proteic killer locus (parDE+) with antisense killer loci (hok+/sok+) had a negligible impact on specific growth rate, maintained high beta-galactosidase expression, and led to a 30 and 190% increase in segregational stability (based on stable generations) as compared to plasmids containing either hok+/sok+ or parDE+ alone, respectively. Use of hok+/sok+ or parDE+ alone with high cloned-gene expression led to ninefold and fourfold increases in the number of stable generations, respectively. Two convenient cloning cassettes have been constructed to facilitate cloning the dual hok+/sok+ parDE+ and hok+/sok+ pnd+ killer systems.     

Genetic mapping of the F plasmid gene that promotes degradation of stable ribonucleic acid in Escherichia coli.

F+ Escherichi coli cells that contain an srnA mutant allele degrade their stable ribonucleic acid (RNA) extensively after RNA synthesis is blocked at 42 degrees C. The relevant gene promoting degradation of stable RNA, srnB+, or its promoter was mapped between 1.7 and 2.8 kilobases on the F plasmid by using deleted F' plasmids and chimeric plasmids composed of pSC101 and fragments of F plasmid.     

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene

In Escherichia coli cells carrying the srnB+ gene of the F plasmid, rifampin, added at 42 degrees C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257-258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784-789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolydigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (Mr 12 000) newly synthesized at 42 degrees C in the presence of rifampin appeared to be the product of the srnB+ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30 degrees C, or the addition of Mg2+ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB+ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB+ gene.     

Mechanism of post-segregational killing: translation of Hok, SrnB and Pnd mRNAs of plasmids R1, F and R483 is activated by 3''-end processing.

The gene systems hok/sok of R1, srnB of F and pnd of R483 mediate plasmid maintenance by killing of plasmid-free segregants. Translation of the very stable mRNAs encoding the killer proteins is regulated by small unstable antisense RNAs. The differential decay rates of the inhibitory antisense RNAs and the mRNAs encoding the killer proteins is the basis for the onset of killer mRNA translation in newborn plasmid-free segregants and the killing of these cells. We have suggested previously that this requires that the killer mRNAs occur in two forms. A translationally inactive form was proposed to be converted into a 3'-truncated, translationally active mRNA. In the presence of the antisense RNA, translation from this killer mRNA should be inhibited. In this communication we present in vivo and in vitro evidence that support this model. The requirement for 3'-processing for killer gene expression is demonstrated. By using in vitro techniques it is shown that full-length Hok mRNA is translationally inactive, whereas a 3'-end truncated version of the Hok mRNA is translationally active. In vitro secondary structure probing suggests that the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of the mok gene and thereby inhibits translation of the mRNA. By inference we conclude that the Pnd and SrnB mRNAs are regulated by a similar mechanism.     

F-plasmid gene srnB+ complements the function of the S gene of phage lambda

Abstract The srnB ⁺ gene located on the F plasmid was assayed for its capacity to facilitate the release from infected cells of phage λ lacking the usual lytic activity. The srnB ⁺ plasmid pOY54, carrying the 1.4–2.5F fragment in the Eco RI- Bam HI fragment of pBR322, induced bacteriolysis and the release of progeny phage of the λcI 857 susS 7 lysogen in the presence of rifampin at 42°C. An srnB 1 mutant plasmid, pOY541, did not promote bacteriolysis. These results suggest that the srnB ⁺ gene of the F plasmid complements the function of the λ S gene in the nonpermissive host strain.     

Translational control and differential RNA decay are key elements regulating postsegregational expression of the killer protein encoded by the parB locus of plasmid R1

The parB locus of plasmid R1, which mediates plasmid stability via postsegregational killing of plasmid-free cells, encodes two genes, hok and sok. The hok gene product is a potent cell-killing protein. The hok gene is regulated at the translational level by the sok gene-encoded repressor, a small anti-sense RNA complementary to the hok mRNA. The hok mRNA is extraordinarily stable, while the sok RNA decays rapidly. The mechanism of postsegregational killing is explained by the following model; the sok RNA molecule rapidly disappears in cells that have lost a parB-carrying plasmid, leading to translation of the stable hok mRNA. Consequently, the Hok protein is synthesized and killing of the plasmid-free cell follows.     

Programmed cell death in bacteria: translational repression by mRNA end-pairing

The hok / sok and pnd systems of plasmids R1 and R483 mediate plasmid maintenance by killing plasmid-free cells. Translation of the exceptionally stable hok and pnd mRNAs is repressed by unstable antisense RNAs. The different stabilities of the killer mRNAs and their cognate repressors explain the onset of translation in plasmid-free cells. The full-length hok and pnd mRNAs are inert with respect to translation and antisense RNA binding. We have previously shown that the mRNAs contain two negative translational control elements. Thus, the mRNAs contain upstream anti-Shine–Dalgarno elements that repress translation by shielding the Shine–Dalgarno ele-ments. The mRNAs also contain fold-back-inhibition elements ( fbi  ) at their 3' ends that are required to maintain the inert mRNA configuration. Using genetic complementation, we show that the 3' fbi elements pair with the very 5' ends of the mRNAs. This pairing sets the low rate of 3' exonucleolytical processing, which is required for the accumulation of an activatable pool of mRNA. Unexpectedly, the hok and pnd mRNAs were found to contain translational activators at their 5' ends (termed tac  ). Thus, the fbi elements inhibit translation of the full-length mRNAs by sequestration of the tac elements. The fbi elements are removed by 3' exonucleolytical processing. Mutational ana-lyses indicate that the 3' processing triggers refolding of the mRNA 5' ends into translatable configurations in which the 5' tac elements base pair with the anti-Shine–Dalgarno sequences.     

Cloning of a 1.1-kb Fragment Including srnB+ Gene in the F Plasmid and Isolation of an srnB Mutant

The srnB⁺ gene, promoting stable RNA degradation at 42 C in the presence of rifampin, was cloned by using pBR322 as a vector; it was located on a 1.1-kilobase (kb) EcoRI/BamHI fragment between 1.4 and 2.5 kb of the F plasmid. The region between 93.3 and 4.0 kb of the F plasmid was physically mapped by using restriction endonucleases EcoRI, HindIII, BamHI, PstI, and SmaI, with reference to a standard HindIII site in IS3. An srnB1 mutant was isolated from a chimeric plasmid, pOY54, after treatment of its DNA with hydroxylamine. The srnB1 allele on the F fragment of the mutant plasmid was recessive to the wild-type allele. Thermal elevation of cell cultures to 39 C was high enough to promote RNA degradation in strain YS12 carrying plasmid pOY54.