RESUMO
In prokaryotes, the centromere is a specialized segment of DNA that promotes the assembly of the segrosome upon binding of the Centromere Binding Protein (CBP). The segrosome structure exposes a specific surface for the interaction of the CBP with the motor protein that mediates DNA movement during cell division. Additionally, the CBP usually controls the transcriptional regulation of the segregation system as a cell cycle checkpoint. Correct segrosome functioning is therefore indispensable for accurate DNA segregation. Here, we combine biochemical reconstruction and structural and biophysical analysis to bring light to the architecture of the segrosome complex in Type III partition systems. We present the particular features of the centromere site, tubC, of the model system encoded in Clostridium botulinum prophage c-st. We find that the split centromere site contains two different iterons involved in the binding and spreading of the CBP, TubR. The resulting nucleoprotein complex consists of a novel double-ring structure that covers part of the predicted promoter. Single molecule data provides a mechanism for the formation of the segrosome structure based on DNA bending and unwinding upon TubR binding.
Assuntos
Centrômero/química , Centrômero/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Sítios de Ligação , Centrômero/metabolismo , Clostridium botulinum/genética , DNA Bacteriano/química , Óperon , Regiões Promotoras Genéticas , Prófagos/genéticaRESUMO
Differential stability of toxins and antitoxins is the key for the conditional activation and function of Toxin-Antitoxin systems. Here we report the evaluation of the action of cell proteases Lon, ClpAP, ClpXP and ClpYQ on the Kis antitoxin and the Kid toxin of the parD TA system of plasmid R1. In vitro analysis shows that Kis antitoxin, but not the Kid toxin, is cleaved specifically by the ClpAP protease. The Kid toxin is not cleaved either by this protease or by any of the others cell proteases tested but in complex with the Kis antitoxin protects the cleavage of this protein in a way that is dependent on the toxin-antitoxin ratio. We further show that this protection is correlated with the inability of the ClpA chaperone to access the Kis antitoxin when in complex with Kid toxin. The stability of the antitoxin greatly increases in vivo in a clpP- background and plasmid maintenance mediated by the parD system, which is dependent on the differential decay of the antitoxin, is reduced to the levels observed in the absence of a functional toxin. The functional implications of these data are further discussed within the frame of the regulation of the parD system and of the available information on the nature of the toxin-antitoxin complexes formed at different toxin-antitoxin ratios.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Plasmídeos/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/genética , Endopeptidase Clp/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Óperon , Plasmídeos/genética , Ligação Proteica , Estabilidade Proteica , ProteóliseRESUMO
Fungal ribotoxins are toxic secreted ribonucleases that cleave a conserved single phosphodiester bond located at the sarcin/ricin loop of the larger rRNA. This cleavage inactivates ribosomes leading to protein biosynthesis inhibition and cell death. It has been proposed that interactions other than those found at the active site of ribotoxins are needed to explain their exquisite specific activity. The study presented shows the ability of a catalytically inactive α-sarcin mutant (H137Q) to bind eukaryotic ribosomes and interfere with in vitro protein biosynthesis. The results obtained are compatible with previous observations that α-sarcin can promote cell death by a mechanism that is independent of rRNA cleavage, expanding the potential set of activities performed by this family of toxins.
Assuntos
Endorribonucleases/farmacologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Animais , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas Fúngicas/genética , Modelos Biológicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/farmacologia , Ligação Proteica , Inibidores da Síntese de Proteínas/metabolismo , Coelhos , Ribossomos/metabolismo , Saccharomyces cerevisiaeRESUMO
The chromosome of the pathogenic Gram-positive bacterium Streptococcus pneumoniae contains between six to 10 operons encoding toxin-antitoxin systems (TAS). TAS are widespread and redundant in bacteria and archaea and their role, albeit still obscure, may be related to important aspects of bacteria lifestyle like response to stress. One of the most abundant TAS is the relBE family, being present in the chromosome of many bacteria and archaea. Because of the high rates of morbility and mortality caused by S. pneumoniae, it has been interesting to gain knowledge on the pneumococcal TAS, among them the RelBE2Spn proteins. Here, we have analyzed the DNA binding capacity of the RelB2Spn antitoxin and the RelB2Spn-RelE2Spn proteins by band-shift assays. Thus, a DNA region encompassing the operator region of the proteins was identified. In addition, we have used analytical ultracentrifugation and native mass spectrometry to measure the oligomerization state of the antitoxin alone and the RelBE2Spn complex in solution bound or unbound to its DNA substrate. Using native mass spectrometry allowed us to unambiguously determine the stoichiometry of the RelB2Spn and of the RelBE2Spn complex alone or associated to its DNA target.
Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Proteínas de Ligação a DNA/química , DNA/química , Streptococcus pneumoniae/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Sequência de Bases , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , UltracentrifugaçãoRESUMO
The coupling between the replication and parD (kis, kid) maintenance modules of R1 has been revisited here by the isolation of a significant collection of conditional replication mutants in the pKN1562 mini-R1 plasmid, and in its derivative, pJLV01, specifically affected in the RNase activity of the Kid toxin. This new analysis aims to identify key factors in this coupling. For this purpose we have quantified and characterized the restriction introduced by parD to isolate conditional replication mutants of this plasmid, a signature of the modular coupling. This restriction depends on the RNase activity of the Kid toxin and it is relieved by either over-expression of the Kis antitoxin or by preventing its degradation by Lon and ClpAP proteases. Based on these data and on the correlation between copy numbers and parD transcriptional levels obtained in the different mutants, it is proposed that a reduction of Kis antitoxin levels in response to inefficient plasmid replication is the key factor for coupling plasmid replication and parD modules.
Assuntos
Proteínas de Bactérias/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Fatores R/genética , Fatores R/metabolismo , Origem de Replicação , Variações do Número de Cópias de DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ordem dos Genes , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Fenótipo , Transativadores/genética , Transativadores/metabolismo , Transcrição GênicaRESUMO
Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a approximately 10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Códon de Terminação , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Mutagênese , Fatores de Terminação de Peptídeos/genéticaRESUMO
The parD operon of Escherichia coli plasmid R1 encodes a toxin-antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid-kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid-Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2-Kis2-Kid2-Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2-Kis2-Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2-Kis2)n complexes repress the kid-kis operon.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regiões Operadoras Genéticas , Plasmídeos/genética , Regiões Promotoras Genéticas , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/metabolismo , Dimerização , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Dados de Sequência Molecular , Subunidades Proteicas/metabolismoRESUMO
The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here, we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We report about the interactions between Kid and Kis that are responsible for neutralization of Kid toxicity and enhance autoregulation of parD transcription. Native macromolecular mass spectrometry data demonstrate that Kid and Kis form multiple complexes. At Kis:Kid ratios equal to or exceeding 1:1, as found in vivo in a plasmid-containing cell, various complexes are present, ranging from Kid(2)-Kis(2) tetramer up to Kis(2)-Kid(2)-Kis(2)-Kid(2)-Kis(2) decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid(2)-Kis(2)-Kid(2) heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the (1)H (15)N HSQC spectra of Kid and Kis, observed when titrating the partner protein, show that the interaction sites of Kid and Kis resemble those within the previously reported MazF(2)-MazE(2)-MazF(2) complex. Furthermore, we demonstrate that Kid(2)-MazE(2) tetramers can be formed via weak interactions involving a limited part of the Kis-binding residues of Kid. The functional roles of the identified Kid-Kis and Kid-MazE interaction sites and complexes in toxin neutralization and repression of transcription are discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Antitoxinas , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Ressonância Magnética Nuclear Biomolecular , Óperon , Plasmídeos/genética , Relação Estrutura-AtividadeRESUMO
The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5' side of the A residue in the nucleotide sequence 5'-UA(A/C)-3' of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2':3'-cyclic phosphate group and a free 5'-OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2'-OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Citotoxinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA/metabolismo , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Domínio Catalítico , Citotoxinas/química , Proteínas de Ligação a DNA/química , Dimerização , Escherichia coli/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Plasmídeos , Ligação Proteica , Conformação Proteica , RNA/químicaRESUMO
Toxin-antitoxin systems were discovered as plasmid auxiliary maintenance cassettes. In recent years, an increasing amount of structural and functional information has become available about the proteins involved, allowing the understanding of bacterial cell growth inhibition by the toxins on a molecular level. A well-studied TA system is formed by the proteins Kid and Kis, encoded by the parD operon of the Escherichia coli plasmid R1. The toxicity of Kid has been related to its endoribonuclease activity, which is counteracted by binding of the antitoxin Kis at the proposed active site. In this review, the structural studies on the Kid-Kis system are compared to those of three related toxin-antitoxin systems: MazF-MazE, CcdB-CcdA and RelE-RelB.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Sequência de Aminoácidos , Antitoxinas , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Fatores R/metabolismo , RNA/metabolismo , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
Toxin-antitoxin (TA) genes were first reported in plasmids and were considered expendable genetic cassettes involved in the stable maintenance of the plasmid replicon by interfering with growth and/or viability of bacteria in which the plasmid was lost. TAs were later found in bacterial chromosomes and also in integrated mobile genetic elements; they were proposed to be involved in the bacterial response to stressful situations. At present, 100s of TAs have been identified and classified in up to six families (I to VI), with those belonging to the type II (constituted by two protein components) being the most studied. Based on well-characterized examples of several type II TAs, we discuss in this review that irrespective of their locations in plasmids or chromosomes, TAs functionally overlap as indicated by: (i) in both locations they can mediate the maintenance of genetic elements to which they are physical linked, and (ii) they can induce persistence or virulence in response to stress situations. Examples of functional confluences in homologous TA systems with different locations are also given. We also consider whether the physiological role of TAs is due to their genetic organization as operons or to their inherent properties, like the short lifespan of the antitoxin component.
RESUMO
Although differing in size, encoded traits, host range, and replication mechanism, both narrow-host-range theta-type conjugative enterobacterial plasmid R1 and promiscuous rolling-circle-type mobilizable streptococcal plasmid pMV158 encode a transcriptional repressor protein, namely CopB in R1 and CopG in pMV158, involved in replication control. The gene encoding CopB or CopG is cotranscribed with a downstream gene that encodes the replication initiator Rep protein of the corresponding plasmid. However, whereas CopG is an auto-repressor that inhibits transcription of the entire copG-repB operon, CopB is expressed constitutively and represses a second, downstream promoter that directs transcription of repA. As a consequence of the distinct regulatory pathways implied by CopB and CopG, these repressor proteins play a different role in control of plasmid replication during the steady state: while CopB has an auxiliary role by keeping repressed the regulated promoter whenever the plasmid copy number is above a low threshold, CopG plays a primary role by acting coordinately with RNAII. Here, we have studied the role of the regulatory circuit mediated by these transcriptional repressors during the establishment of these two plasmids in a new host cell, and found that excess Cop repressor molecules in the recipient cell result in a severe decrease in the frequency and/or the velocity of appearance of transformant colonies for the cognate plasmid but not for unrelated plasmids. Using the pMV158 replicon as a model system, together with highly sensitive real-time qPCR and inverse PCR methods, we have also analyzed the effect of CopG on the kinetics of repopulation of the plasmid in Streptococcus pneumoniae. We show that, whereas in the absence of CopG pMV158 repopulation occurs mainly during the first 45 min following plasmid transfer, the presence of the transcriptional repressor in the recipient cell severely impairs the replicon repopulation and makes the plasmid replicate at approximately the same rate as the chromosome at any time after transformation, which results in maximal plasmid loss rate in the absence of selection. Overall, these findings indicate that unrepressed activity of the Cop-regulated promoter is crucial for the successful colonization of the recipient bacterial cells by the plasmid.
RESUMO
Since the ban gene of bacteriophage P1 suppresses a number of conditionally lethal dnaB mutations in Escherichia coli, it was assumed that Ban protein is a DNA helicase (DnaB analogue) that can substitute for DnaB in the host replication machinery. We isolated and sequenced the ban gene, purified the product, and analysed the function of Ban protein in vitro and in vivo. Ban hydrolyses ATP, unwinds DNA and forms hexamers in the presence of ATP and magnesium ions. Since all existing conditionally lethal dnaB strains bear DnaB proteins that may interfere with the protein under study, we constructed a dnaB null strain by using a genetic set-up designed to provoke the conditional loss of the entire dnaB gene from E.coli cells. This novel tool was used to show that Ban restores the viability of cells that completely lack DnaB at 30 degrees C, but not at 42 degrees C. Surprisingly, growth was restored by the dnaB252 mutation at a temperature that is restrictive for ban and dnaB252 taken separately. This indicates that Ban and DnaB are able to interact in vivo. Complementary to these results, we demonstrate the formation of DnaB-Ban hetero-oligomers in vitro by ion exchange chromatography. We discuss the interaction of bacterial proteins and their phage-encoded analogues to fulfil functions that are essential to phage and host growth.
Assuntos
Proteínas de Bactérias , Bacteriófago P1/genética , DNA Helicases/metabolismo , Escherichia coli/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bacteriófago P1/enzimologia , DNA Helicases/genética , DNA Viral/química , DNA Viral/genética , Dimerização , DnaB Helicases , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Ligação Proteica , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 A crystal structure of Kid reveals a 2-fold symmetric dimer that closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli F plasmid despite the lack of any notable sequence similarity. Analysis of nontoxic mutants of Kid suggests a target interaction interface associated with toxicity that is in marked contrast to that proposed for CcdB. A possible region for interaction of Kid with the antitoxin is proposed that overlaps with the target binding site and may explain the mode of antitoxin action.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/farmacologia , Escherichia coli/química , Plasmídeos , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Bacterial virulence relies on a delicate balance of signals interchanged between the invading microbe and the host. This communication has been extensively perceived as a battle involving harmful molecules produced by the pathogen and host defenses. In this review, we focus on a largely unexplored element of this dialogue, as are toxin-antitoxin (TA) systems of the pathogen. TA systems are reported to respond to stresses that are also found in the host and, as a consequence, could modulate the physiology of the intruder microbe. This view is consistent with recent studies that demonstrate a contribution of distinct TA systems to virulence since their absence alters the course of the infection. TA loci are stress response modules that, therefore, could readjust pathogen metabolism to favor the generation of slow-growing or quiescent cells 'before' host defenses irreversibly block essential pathogen activities. Some toxins of these TA modules have been proposed as potential weapons used by the pathogen to act on host targets. We discuss all these aspects based on studies that support some TA modules as important regulators in the pathogen-host interface.
Assuntos
Antitoxinas/metabolismo , Bactérias/patogenicidade , Toxinas Bacterianas/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , VirulênciaRESUMO
Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50-90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdABST and vapBC2ST). The TA module ccdABST has previously been shown to contribute to pSLT plasmid stability and vapBC2ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdABST encodes an inactive CcdBST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdABST variant containing a single mutation (R99W) that restores the toxicity of CcdBST. The "activation" of CcdBST (R99W) did not increase pSLT stability by ccdABST. In contrast, ccdABST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdABST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdAST antitoxin more than on its toxic activity.
RESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Replicação do DNA , Fatores R/genética , Fatores R/metabolismo , Origem de Replicação , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Western Blotting , Variações do Número de Cópias de DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Toxin-antitoxin (TA) modules contribute to the generation of non-growing cells in response to stress. These modules abound in bacterial pathogens although the bases for this profusion remain largely unknown. Using the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as a model, here we show that a selected group of TA modules impact bacterial fitness inside eukaryotic cells. We characterized in this pathogen twenty-seven TA modules, including type I and type II TA modules encoding antisense RNA and proteinaceous antitoxins, respectively. Proteomic and gene expression analyses revealed that the pathogen produces numerous toxins of TA modules inside eukaryotic cells. Among these, the toxins HokST, LdrAST, and TisBST, encoded by type I TA modules and T4ST and VapC2ST, encoded by type II TA modules, promote bacterial survival inside fibroblasts. In contrast, only VapC2ST shows that positive effect in bacterial fitness when the pathogen infects epithelial cells. These results illustrate how S. Typhimurium uses distinct type I and type II TA modules to regulate its intracellular lifestyle in varied host cell types. This function specialization might explain why the number of TA modules increased in intracellular bacterial pathogens.
Assuntos
Antitoxinas/imunologia , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Células Eucarióticas/microbiologia , Interações Hospedeiro-Patógeno , Salmonella/fisiologia , Antitoxinas/genética , Toxinas Bacterianas/genética , Análise por Conglomerados , Biologia Computacional/métodos , Células Epiteliais/microbiologia , Fibroblastos/microbiologia , Loci Gênicos , Genoma Bacteriano , Humanos , Salmonella typhimurium/fisiologiaRESUMO
The mazEF (chpA) toxin-antitoxin system of Escherichia coli is involved in the cell response to nutritional and antibiotic stresses as well as in bacterial-programmed cell death. Valuable information on the MazF toxin was derived from the determination of the crystal structure of the MazE/MazF complex and from in vivo data, suggesting that MazF promoted ribosome-dependent cleavage of messenger RNA. However, it was concluded from recent in vitro analyses using a MazF-(His6) fusion protein that MazF was an endoribonuclease that cleaved messenger RNA specifically at 5'-ACA-3' sites situated in single-stranded regions. In contrast, our work reported here shows that native MazF protein cleaves RNA at the 5' side of residue A in 5'-NAC-3' sequences (where N is preferentially U or A). MazF-dependent cleavage occurred at target sequences situated either in single- or double-stranded RNA regions. These activities were neutralized by a His6-MazE antitoxin. Although essentially consistent with previous in vivo reports on the substrate specificity of MazF, our results strongly suggest that the endoribonuclease activity of MazF may be modulated by additional factors to cleave messenger and other cellular RNAs.
Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , Animais , Antitoxinas/metabolismo , Toxinas Bacterianas/química , Sequência de Bases , Sítios de Ligação , Dicroísmo Circular , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , RNA Bacteriano/química , Coelhos , Reticulócitos/metabolismo , Especificidade por Substrato , UltracentrifugaçãoRESUMO
We report the identification and genetic analysis of mutants in the antitoxin of the parD (kis, kid) killer system of plasmid R1. Missense mutants placed at codons 10, 11, 12 and 18 maintained the antitoxin activity of Kis, but not the ability of this protein to co-regulate the parD system together with the Kid toxin. Deletion of the last 33 amino acids of Kis inactivated the antitoxin activity of the protein and reduced substantially, but not completely, its regulatory activity. These results define two functional regions in Kis: an amino-terminal region which is specifically involved in regulation, and a carboxy-terminal region of the protein, which is important both for its regulatory and antitoxin activities.