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K Gerdes

Publications and source records attributed to K Gerdes.

At least 19 recordsLinked to original sources
PubMed1996

The centromere-like parC locus of plasmid R1.

The parA partitioning system of plasmid R1 consists of three components: the cis-acting centromere-like parC locus, and two proteins, ParM and ParR. The parC locus contains two sets of five direct repeats (iterons) to which the ParR protein binds. The parA promoter is located in the core region between the two sets of iterons. Mini-R1 replicons carrying parC are stabilized by the simultaneous presence of ParM and ParR. The parC locus present on a co-resident plasmid leads to instability of the mini-R1 replicon (incompatibility). Here we present a genetic analysis of the stability and incompatibility phenotypes associated with parC. We show that all 10 iterons are required for maximum stabilization and incompatibility. Replacement of the core promoter region between the repeats by a foreign promoter region did not reduce stabilization. Thus, the only structural components in parC seem to be the two sets of iterons. The parA promoter, P parA, is repressed by ParR. We show that all 10 iterons are required for full repression of the promoter. The activity of the promoter was influenced by sequences located outside the core region. An A-rich region located upstream of the -35 element of PparA was found to increase promoter activity. The region encoding the parA mRNA leader region also strongly influenced the expression level of PparA- lacZ fusions. We show that this high expression (hex) element is a transcriptional antiterminator that prevents Rho-dependent termination.

Bacterial Proteins↗
PubMed1995

Mechanism of post-segregational killing by hok-homologue pnd of plasmid R483: two translational control elements in the pnd mRNA.

The pnd system of plasmid R483 mediates plasmid stabilization by killing of plasmid-free cells. The pnd mRNA is very stable and can be translated into PndA protein, a cell toxin which kills the cells from within by damaging the cell membrane. Translation of the pnd mRNA is inhibited by the PndB antisense, a small labile RNA of 63 nt. The rapid decay of the PndB antidote leads to onset of PndA synthesis in plasmid-free segregants or after addition of rifampicin. Surprisingly however, the full-length pnd mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was found to be active. We have therefore suggested previously, that the 3'-end of the full-length pnd mRNA encodes a fold-back inhibitory sequence (fbi), which prevents its translation. Here we present an analysis of the metabolism of the pnd mRNAs. A mutational analysis shows that single point mutations in the fbi motif results in more rapid truncation. The fbi mutations could not be complemented by second-site mutations in either of the pndA or pndC Shine-Dalgarno (SD) elements. Surprisingly, mutations in the pndC SD element also lead to a more rapid truncation. The effect of these latter mutations was, however, complemented by mutations in a proposed anti-SD element upstream of the pndC SD. Mutations in the anti-SD element were lethal. These results show, that the pnd mRNA contains two negative control elements, one located in its very 3'-end (fbi), and one located just upstream of the pndC SD region (the anti-SD element). These observations add to the complexity of the induction scheme previously proposed to explain activation of pndA expression in plasmid-free cells: In addition to its negative effect of translation, the fbi structure also maintains a reduced processing rate in the 3'-end of the mRNA. This permits the accumulation of a reservoir of pnd mRNA, which can be activated by 3'-end processing in plasmid-free cells. The anti-SD may prevent translation of the pnd mRNA during transcription, thus preventing detrimental synthesis of toxin.

Bacterial Proteins↗
PubMed1995

Mechanism of post-segregational killing: secondary structure analysis of the entire Hok mRNA from plasmid R1 suggests a fold-back structure that prevents translation and antisense RNA binding.

The hok/sok system of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The Hok mRNA is very stable and can be translated into Hok killer protein. Translation of the Hok mRNA is inhibited by the small unstable Sok antisense RNA. Translation of hok is coupled to an overlapping reading frame termed mok. Translation of mok is tightly regulated by Sok RNA, and Sok RNA thus regulates hok translation indirectly through mok. The rapid decay of Sok RNA explains the onset of Hok synthesis in newborn plasmid-free segregants. However, a second control level is superimposed on this simple induction scheme, since the full-length Hok mRNA was found to be translationally inactive whereas a 3'-end truncated version of it was active. We have therefore previously suggested, that the 3'-terminal region of the full-length Hok mRNA encodes an element which prevents its translation. This element was termed fbi (fold-back inhibition). Here we describe the in vitro secondary structure of the entire Hok mRNA. Our results suggest a closed structure in which the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of mok. This structure explains why full-length Hok mRNA is translationally silent. The proposed structure was further supported by results obtained using mutations in the 3'-end fbi element. These "structure closing" mutations affected the structure much further upstream in the mok translational initiation region and concomitantly prevented antisense RNA binding to the same region of the mRNA. These results lend further support to the induction model that explains onset of Hok mRNA translation in plasmid-free segregants. The most important regulatory element in this model is the FBI structure formed between the 3'-end and the mok translational initiation region. This structure renders Hok mRNA translationally inactive and prevents antisense RNA binding, thus allowing the accumulation of a pool of mRNA which, by slow 3'-end processing, is activated in plasmid-free segregants, eventually leading to the elimination of these cells.

Bacterial Proteins↗
PubMed1995

Programmed cell death in bacteria: proteic plasmid stabilization systems.

Bacterial plasmids are stabilized by a number of different mechanisms. Here we describe the molecular aspects of a group of plasmid-encoded gene systems called the proteic killer gene systems. These systems mediate plasmid maintenance by selectively killing plasmid-free cells (post-segregational killing or plasmid addiction). The group includes ccd of F, parD/pem of R1/R100, parDE of RP4/RK2, and phd/doc of P1. All of these systems encode a stable toxin and an unstable antidote. The antidotes prevent the lethal action of their cognate toxins by forming tight complexes with them. The antidotes are degraded by cellular proteases. Thus, the different decay rates of the toxins and antidotes seem to be the molecular basis of toxin activation in plasmid-free cells. The operons encoding the toxins and antidotes are autoregulated at the level of transcription either by a complex formed by the toxins and the cognate antidotes or by the antidote alone. The cellular targets of the killer proteins have been determined to be DNA gyrase in the case of ccd of F and DnaB in the case of parD of R1. Surprisingly, the Escherichia coli chromosome encodes at least two of these peculiar gene systems.

Antidotes↗
PubMed1995

Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1.

A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants.

Bacterial Proteins↗
PubMed1994

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.

Aminoglycosides↗
PubMed1994

Mechanism of post-segregational killing: Sok antisense RNA interacts with Hok mRNA via its 5'-end single-stranded leader and competes with the 3'-end of Hok mRNA for binding to the mok translational initiation region.

The hok/sok system of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNA species, Hok mRNA and Sok antisense RNA. The lethal expression of hok is inhibited post-transcriptionally by the 67 nt Sok-RNA. In this paper, we analyse the secondary structure of Sok-RNA and the binding of Sok-RNA to Hok mRNA in vitro. The reaction between the two RNAs leads to the formation of a complete duplex in which Sok-RNA is hybridized over its entire length to Hok mRNA. The second-order rate constant of duplex formation was determined to be approximately 1 x 10(5) M-1s-1. Mutations in the 5'-end single-stranded leader of Sok-RNA severely reduced the binding rate to wt Hok mRNA, whereas loop mutations in Sok-RNA had no such effect. The reduced binding rates were paralleled by abolished in vivo regulatory properties. These results suggest that, unlike in other well-characterized antisense/target RNA systems, the initial recognition reaction between Sok-RNA and Hok mRNA takes place between the single-stranded 5'-end of Sok-RNA and the complementary region in Hok mRNA, without the involvement of an antisense loop in the initial binding step. Furthermore, the finding that Sok-RNA competes with the 3'-end of full-length Hok mRNA for binding to the mok translational initiation region adds to the complexity of killer gene regulation.

Bacterial Proteins↗
PubMed1994

Partitioning of plasmid R1. Ten direct repeats flanking the parA promoter constitute a centromere-like partition site parC, that expresses incompatibility.

The parA partitioning system of plasmid R1 consists of three different components: the cis-acting centromere-like parC site, and the two trans-acting proteins ParM and ParR. These three components are contained within a region of 1.6 kb. The parC site is located upstream of the two genes, parM and parR, which are expressed as an operon from the parA promoter. The parC site contains an array of ten 11 base-pair direct repeats, organized in two sets of five repeats flanking the parA core promoter sequences. Deletions and point mutations were introduced in the parA locus, resulting in partially stable and unstable plasmids. An analysis of these parA- plasmids showed that ParM and ParR are transacting. The 160 bp minimal parC region contained sufficient in cis information for efficient trans-complementation. Both proteins were required for maximal stabilization of a parC+ mini-R1 plasmid, although ParR alone, donated either in cis or in trans, yielded partial stabilization. Plasmids that overexpressed ParR caused destabilization of a co-resident parA+ plasmid, whereas overexpression of ParM had no such effect. The parC site exerted incompatibility (incA) at high but not at low copy number. Likewise, the entire parA system exerted incompatibility in a copy number-dependent fashion, and stronger than the incompatibility expressed by parC alone.

Base Sequence↗
PubMed1994

Partitioning of plasmid R1. The parA operon is autoregulated by ParR and its transcription is highly stimulated by a downstream activating element.

The parA partitioning system of plasmid R1 mediates efficient stabilization of R1 and F-derived replicons. The parA system is encoded by a continuous DNA segment of approximately 1600 base-pairs and consists of three components. Two adjacent genes, parM and parR, coding for the trans-acting proteins ParM and ParR, and the cis-acting parC site. The centromere-like parC site is located upstream of parM and parR and contains the parA promoter. The parM and parR genes are co-transcribed as an operon from the parA promoter. The 5' end of the parA encoded transcript was mapped to the center of the parC region at +115. The -10 and -35 core promoter sequences are flanked by the two sets of five direct repeats in parC (the ParR boxes). The parA promoter was found to be negatively regulated by the parR gene product, whereas the parM gene product seemingly was not involved in the regulation. Surprisingly, a region downstream of the parA promoter enhanced transcription from the promoter many-fold (30 to 50-fold). The parC site titrated the ParR protein, suggesting that the ParR protein interacts directly with the parC site. Using an engineered parA system we found that the parC site could be complemented in cis by the parM and parR genes. Furthermore, the proper function of the parC site was highly dependent on the expression level of ParM and ParR. The incompatibility associated with the parC site could not be suppressed by overexpression of the ParM and ParR proteins. Based on these results we suggest a novel partition model involving pairing of newly replicated plasmid molecules.

Bacterial Proteins↗
PubMed1992

Mechanism of killer gene activation. Antisense RNA-dependent RNase III cleavage ensures rapid turn-over of the stable hok, srnB and pndA effector messenger RNAs.

The hok/sok, srnB and pnd systems of plasmids R1, F and R438 mediate plasmid maintenance by killing plasmid-free segregants. The systems encode exceptionally stable full-length mRNAs that code for potent cell toxins that kill the cells from within. The systems also produce truncated mRNAs whose appearance is correlated with killing activity. The truncated mRNAs are shortened by 35 to 70 nucleotides in the 3' ends, but have the same 5' ends as the full-length transcripts. Translation of the stable killer mRNAs is regulated by unstable antisense RNAs that are complementary to the leader regions of the full-length and truncated mRNAs. We show here, that both the presence of the antisense RNA and of the host enzyme RNase III is required for rapid cleavage of the truncated mRNAs, and we map the cleavage point in the Hok mRNA in vitro and in vivo to be located between nucleotides +245 and +246. The RNase III cleavage products of the Hok mRNA were found to be very unstable in vivo. Thus, RNase III cleavage seems to be the initial event leading to decay of the killer mRNAs. In an rnc- strain, the truncated mRNA species were found in steady-state cells. This observation indicates that the truncated mRNAs are formed constitutively and independently of the presence of the antisense RNAs. Thus, the antisense RNAs prevent the accumulation of the truncated mRNAs solely by mediating their rapid hydrolysis by RNase III. Furthermore, the generation of the truncated killer mRNAs in the rnc- host indicate that RNase III is dispensable for induction of the killer gene systems. Based on these and on observations obtained previously, we present a molecular model that explains the activation of the killer mRNAs in plasmid-free segregants and after addition of rifampicin.

Base Sequence↗
PubMed1992

Mechanism of post-segregational killing by the hok/sok system of plasmid R1. Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene.

The hok/sok locus of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNAs, Hok mRNA and Sok antisense RNA. Hok mRNA encodes the Hok killer protein of 52 amino acid residues. Expression of hok is regulated post-transcriptionally by Sok antisense RNA. Killing of plasmid-free daughter-cells by the hok/sok system is accomplished through differential decay of the Hok and Sok-RNAs: Hok mRNA is very stable while Sok-RNA decays rapidly, thus leading to derepression of Hok mRNA translation in plasmid-free segregants, ensuring a rapid and selective killing of these cells. Sok antisense RNA is complementary to the leader region of the Hok mRNA. However, the region of complementarity does not overlap with the hok Shine-Dalgarno sequence. Thus, Sok-RNA regulates hok translation indirectly by an as yet unknown mechanism. We show here that Sok antisense RNA regulates the translation of another reading frame located in the hok/sok locus. This new reading frame, which overlaps with almost the entire hok gene, was denoted mok (mediation of killing). Point-mutations that prevent mok translation through the hok translational initiation region abolish efficient expression of hok. Furthermore, these mutations abolish the Sok-RNA-mediated control of hok gene expression. Hence, the antisense-RNA-mediated regulation of the hok gene seems to occur via translational coupling between the hok and mok reading-frames.

Base Sequence↗
PubMed1991

The rifampicin-inducible genes srnB from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by killing 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.

Base Sequence↗
PubMed1991

The kis and kid genes of the parD maintenance system of plasmid R1 form an operon that is autoregulated at the level of transcription by the co-ordinated action of the Kis and Kid proteins.

Stability mediated by the parD system of plasmid R1 is modulated by a killer protein, Kid, and by an antagonist of this function, Kis. Determination of the 5' ends of ParD transcripts, revealed that the genes coding for these proteins are transcribed from a single promoter. Analysis of the 3' end of the ParD RNAs indicated the existence of two transcripts: one of them coding for the Kis and Kid proteins, and the other coding only for Kis. Analysis of the effects of parD+ and kis+ recombinants on the beta-galactosidase levels expressed by different transcriptional and translational parD-lacZ fusions, and on the ParD RNA levels determined by a derepressed parD mutant, indicated that the Kis and Kid proteins repress coordinately the parD system at the transcriptional level. We discuss the relevance of these results in terms of the activities of the Kis and Kid proteins and in the context of the stabilization mediated by parD.

Bacterial Proteins↗
PubMed1990

Mechanism of post-segregational killing by the hok/sok system of plasmid R1: sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid-free cells.

The hok/sok system of plasmid R1, which mediates plasmid stabilization via killing of plasmid-free segregants, encodes two genes: hok and sok. The hok gene product is a potent cell-killing protein. The expression of hok is regulated post-transcriptionally by the sok gene-encoded repressor, an antisense RNA complementary to the hok mRNA leader region. We show here that the hok mRNA is very stable, while the sok RNA decays rapidly. We also observe a new hok mRNA species which is 70 nucleotides shorter in the 3'-end than the full-length hok transcript. The appearance of the truncated hok mRNA was found to be regulated by the sok antisense RNA. Furthermore, the presence of the truncated hok mRNA was found to be correlated with efficient expression of the Hok protein. On the basis of these findings, we propose an extended model in order to explain the killing of plasmid-free segregants by the hok/sok system.

Base Sequence↗
PubMed1990

The hok killer gene family in gram-negative bacteria.

The seven members of the hok killer gene family in Gram-negative bacteria are described here. The members of this gene family have been sequenced and include hok/sok from plasmid R1, flm and srnB from plasmid F, pnd from plasmids R483 and R16, and gef and relF, which are located on the Escherichia coli chromosome. The killer proteins encoded by these loci are highly toxic polypeptides of 50 to 52 amino acids. The proteins kill the cells from the inside by interfering with a vital function in the cell membrane. On the basis of their relatedness, the killer proteins and their corresponding loci are divided into four subfamilies. The members of one subfamily, hok/sok and flm, mediate plasmid maintenance by killing plasmid-free cells. The pnd and srnB subfamilies were discovered through their abilities to cause membrane damage and degradation of stable RNA. gef and relF, which constitute the chromosomal subfamily, were found because of their sequence similarity at the DNA and protein levels with other members of the hok gene family. However, no function has been described for the proteins belonging to this subfamily. Although the four subfamilies are distantly related in terms of DNA and protein sequence similarity, the overall genetic organization of the different loci has been well conserved during evolution. The expression of all of the members of the hok gene family is regulated post-transcriptionally. Thus, the expression of the hok and flm genes is regulated by small antisense RNAs that inhibit the translation of the stable hok and flm mRNAs. On the basis of structural and functional similarities, we suggest that each of the related plasmid-encoded killer genes is regulated by antisense RNAs. The conservation of this widespread gene family in Gram-negative bacteria suggests that the genes are important to the genomes that carry them.

Amino Acid Sequence↗
PubMed1988

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.

Base Sequence↗
PubMed1987

Genetic analysis of the parB+ locus of plasmid R1.

Plasmid R1 in Escherichia coli carries two loci which independently contribute to the stable maintenance of the plasmid. A genetic analysis of one of these, parB+, was carried out, and it was shown that the minimal region exerting stabilizing activity comprises at most 580 bp. The nucleotide sequence of the parB+ locus was determined, and indicated the presence of two genes, of which one probably codes for a 52 amino acid polypeptide, whereas the other gene product may be an untranslated RNA. These suggestions, based on the nucleotide sequence information, were supported by gene expression studies employing lac fusions. An incompatibility phenotype connected to parB+ was localized to that part of the 580 bp parB+ region which seems to encode the untranslated RNA.

Base Sequence↗