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Upon release from the stable complex formed with its antitoxin VapB, the toxin VapC (MvpT) of the Gram-negative pathogen Shigella flexneri is capable of globally down-regulating translation by specifically cleaving initiator tRNA(fMet) in the anticodon region. Recombinant Shigella flexneri VapC(D7A) harbouring an active-site mutation was overexpressed in Escherichia coli, purified to homogeneity and crystallized by the vapour-diffusion technique. A preliminary X-ray crystallographic analysis shows that the crystals diffracted to at least 1.9 Å resolution at a synchrotron X-ray source and belonged to the trigonal space group in the hexagonal setting, H3, with unit-cell parameters a = b = 120.1, c = 52.5 Å, α = β = 90, γ = 120°. The Matthews coefficient is 2.46 Å(3) Da(-1), suggesting two molecules per asymmetric unit and corresponding to a solvent content of 50.0%.

Many toxin-antitoxin operons are regulated by the toxin/antitoxin ratio by mechanisms collectively coined "conditional cooperativity". Toxin and antitoxin form heteromers with different stoichiometric ratios, and the complex with the intermediate ratio works best as a transcription repressor. This allows transcription at low toxin level, strong repression at intermediate toxin level, and then again transcription at high toxin level. Such regulation has two interesting features; firstly, it provides a non-monotonous response to the concentration of one of the proteins, and secondly, it opens for ultra-sensitivity mediated by the sequestration of the functioning heteromers. We explore possible functions of conditional regulation in simple feedback motifs, and show that it can provide bistability for a wide range of parameters. We then demonstrate that the conditional cooperativity in toxin-antitoxin systems combined with the growth-inhibition activity of free toxin can mediate bistability between a growing state and a dormant state.

The highly persistent and often lethal human pathogen, Mycobacterium tuberculosis contains at least 88 toxin-antitoxin genes. More than half of these encode VapC PIN domain endoribonucleases that inhibit cell growth by unknown mechanisms. Here we show that VapC20 of M. tuberculosis inhibits translation by cleavage of the Sarcin-Ricin loop (SRL) of 23S ribosomal RNA at the same position where Sarcin and other eukaryotic ribotoxins cleave. Toxin-inhibited cells can be rescued by the expression of the antitoxin, thereby raising the possibility that vapC20 contributes to the extreme persistence exhibited by M. tuberculosis. VapC20 cleavage is inhibited by mutations in the SRL that flank the cleavage site but not by changes elsewhere in the loop. Disruption of the SRL stem abolishes cleavage; however, further mutations that restore the SRL stem structure restore cleavage, revealing that the structure rather than the exact sequence of the SRL is important for this activity.

Many toxin-antitoxin (TA) loci are known to strongly repress their own transcription. This auto-inhibition is often called 'conditional cooperativity' as it relies on cooperative binding of TA complexes to operator DNA that occurs only when toxins are in a proper stoichiometric relationship with antitoxins. There has recently been an explosion of interest in TA systems due to their role in bacterial persistence, however the role of conditional cooperativity is still unclear. We reveal the biological function of conditional cooperativity by constructing a mathematical model of the well studied TA system, relBE of Escherichia coli. We show that the model with the in vivo and in vitro established parameters reproduces experimentally observed response to nutritional stress. We further demonstrate that conditional cooperativity stabilizes the level of antitoxin in rapidly growing cells such that random induction of relBE is minimized. At the same time it enables quick removal of free toxin when the starvation is terminated.

Innate immune cells respond to microbial invaders using pattern recognition receptors that detect conserved microbial patterns. Among the cellular processes stimulated downstream of pattern recognition machinery is the initiation of autophagy, which plays protective roles against intracellular microbes. We have shown recently that Dictyostelium discoideum, which takes up bacteria for nutritive purposes, may employ pattern recognition machinery to respond to bacterial prey, as D. discoideum cells upregulate bactericidal activity upon stimulation by lipopolysaccharide (LPS). Here we extend these findings, showing that LPS treatment leads to induction of autophagosomal maturation in cells responding to the bacteria Staphylococcus aureus. Cells treated with the autophagy-inducing drug rapamycin clear internalized bacteria at an accelerated rate, while LPS-enhanced clearance of bacteria is reduced in cells deficient for the autophagy-related genes atg1 and atg9. These findings link microbial pattern recognition with autophagy in the social amoeba D. discoideum., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Toxin-antitoxin (TA) loci encode inhibitors of translation, replication or cell wall synthesis and are common elements of prokaryotic plasmids and chromosomes. Ten TA loci of Escherichia coli K-12 encode mRNases that cumulatively contribute to persistence (multidrug tolerance) of the bacterial cells. The mechanisms underlying induction and reversion of the persistent state are not yet understood. The vapBC operon of Salmonalla enterica serovar Typhimurium LT2 encodes VapC, a tRNase that reversibly inhibits translation by site-specific cleavage of tRNA(fMet). VapB is an antitoxin that interacts with and neutralizes VapC via its C-terminal tail and regulate TA operon transcription via its N-terminal DNA binding domain that recognize operators in the vapBC promoter region. We show here that transcription of the vapBC operon of S. enterica is controlled by a recently discovered regulatory theme referred to as 'conditional cooperativity': at low T/A ratios, the TA complex binds cooperatively to the promoter region and represses TA operon transcription whereas at high T/A ratios, the excess toxin leads to destabilization of the TA-operator complex and therefore, induction of transcription. We present evidence that an excess of VapC toxin leads to operator complex destabilization by breaking of toxin dimers.

Bacterial persistence is caused by the presence of rare, slowly growing bacteria among populations of rapidly growing cells. The slowly growing bacteria are tolerant of antibiotics and other environmental insults, whereas their isogenic, rapidly growing siblings are sensitive. Recent research has shown that persistence of the model organism Escherichia coli depends on toxin-antitoxin (TA) loci. Deletion of type II TA loci reduces the level of persistence significantly. Lon protease but no other known ATP-dependent proteases is required for persistence. Polyphosphate and (p)ppGpp also are required for persistence. These observations led to the proposal of a simple and testable model that explains the persistence of E. coli. It is now important to challenge this model and to test whether the persistence of pathogenic bacteria also depends on TA loci.

Bacterial cell division relies on the formation and contraction of the Z ring, coordinated and regulated by a dynamic protein complex called the divisome. The cell division factor ZapA interacts directly with FtsZ and thereby increases FtsZ protofilament association and Z-ring stability. Here, we investigated ZapB interaction with ZapA and its effect on Z-ring formation and FtsZ protofilament bundling. The combination of the ftsZ84 allele that encodes an FtsZ variant that polymerizes inefficiently with a zapB null mutant resulted in a synthetic defective phenotype. Overproduction of ZapA led to the formation of aberrant FtsZ helical structures and delocalization of ZapB. The N-terminal end of ZapB was essential for ZapB-ZapA interaction, and amino acid changes close to the N terminus of ZapB exhibited reduced interaction with ZapA. Sedimentation assays showed that ZapB interacts strongly with ZapA and reduces ZapA's interaction with FtsZ in vitro. The morphology of the structures formed by ZapA and ZapB together was similar to the cables formed by ZapB in the presence of CaCl(2), a known ZapB bundling agent. The in vivo and in vitro data support a model in which ZapA interacts strongly with ZapB and the ZapA-ZapB interaction is favored over ZapA-FtsZ.

Toxin-antitoxin (TA) loci are common in archaea and prokaryotes and allow cells to rapidly adapt to changing environmental conditions through release of active regulators of metabolism. Many toxins are endonucleases that target cellular mRNA and tRNAs, while the antitoxins tightly wrap around the toxins to inhibit them under normal circumstances. The antitoxins also bind to operators in the promoter regions of the cognate TA operon and thereby regulate transcription. For enteric vapBC TA loci, the VapC toxins specifically cleave tRNA(fMet) and thus down-regulate protein synthesis. Here, we describe the crystal structure of the intact Shigella flexneri VapBC TA complex, determined to 2.7 Å resolution. Both in solution and in the crystal structure, four molecules of each protein combine to form a large and globular hetero-octameric assembly with SpoVT/AbrB-type DNA-binding domains at each end and a total molecular mass of about 100 kDa. The structure gives new insights into the inhibition of VapC toxins by VapB and provides the molecular basis for understanding transcriptional regulation through VapB dimerization., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Prokaryotic chromosomes and plasmids can be actively segregated by partitioning (par) loci. The common ParA-encoding par loci segregate plasmids by arranging them in regular arrays over the nucleoid by an unknown mechanism. Recent observations indicate that ParA moves plasmids and chromosomes by a pulling mechanism. Even though ParAs form filaments in vitro it is not known whether similar structures are present in vivo. ParA of P1 forms filaments in vitro at very high concentrations only and filament-like structures have not been observed in vivo. Consequently, a 'diffusion-ratchet' mechanism was suggested to explain plasmid movement by ParA of P1. We compare this mechanism with our previously proposed filament model for plasmid movement by ParA. Remarkably, ParA homologues have been discovered to arrange subcellular structures such as carboxysomes and chemotaxis sensory receptors in a regular manner very similar to those of the plasmid arrays., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

2,6-Diazaheptatrienyl metal compounds 6(-)K(+) are easily accessible from the corresponding diimines 6 by deprotonation using KO-t-Bu as base. According to quantum chemical calculations, they are, in comparison to other isomeric species with nitrogen atoms in other positions, highly reactive intermediates, which undergo in dilute solution at 50 °C ring closure reactions to form 3-aminoindole derivatives 8/10. In contrast, in more concentrated solution at room temperature, the formation of 14-membered macrocyclces 13 as a result of formal dimerization is observed. The 3-aminoindole derivatives obtained in this work possess rare substitution patterns, and the macrocyclic compounds are essentially unknown. Two-fold vinylogous derivatives 7 give rise to tricyclic systems with δ-carboline backbone 12. The experimental results are interpreted using high-level DFT calculations with regard to the possible reaction mechanism and the nature of the transition state of the five-membered ring formation. The molecular structures in the solid state of all types of compounds were elucidated by X-ray diffraction.

Bacteria form persisters, individual cells that are highly tolerant to different types of antibiotics. Persister cells are genetically identical to nontolerant kin but have entered a dormant state in which they are recalcitrant to the killing activity of the antibiotics. The molecular mechanisms underlying bacterial persistence are unknown. Here, we show that the ubiquitous Lon (Long Form Filament) protease and mRNA endonucleases (mRNases) encoded by toxin-antitoxin (TA) loci are required for persistence in Escherichia coli. Successive deletion of the 10 mRNase-encoding TA loci of E. coli progressively reduced the level of persisters, showing that persistence is a phenotype common to TA loci. In all cases tested, the antitoxins, which control the activities of the mRNases, are Lon substrates. Consistently, cells lacking lon generated a highly reduced level of persisters. Moreover, Lon overproduction dramatically increased the levels of persisters in wild-type cells but not in cells lacking the 10 mRNases. These results support a simple model according to which mRNases encoded by TA loci are activated in a small fraction of growing cells by Lon-mediated degradation of the antitoxins. Activation of the mRNases, in turn, inhibits global cellular translation, and thereby induces dormancy and persistence. Many pathogenic bacteria known to enter dormant states have a plethora of TA genes. Therefore, in the future, the discoveries described here may lead to a mechanistic understanding of the persistence phenomenon in pathogenic bacteria.

Eukaryotic PIN (PilT N-terminal) domain proteins are ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. The majority of prokaryotic PIN-domain proteins are encoded by the abundant vapBC toxin--antitoxin loci and inhibit translation by an unknown mechanism. Here we show that enteric VapCs are site-specific endonucleases that cleave tRNA(fMet) in the anticodon stem-loop between nucleotides +38 and +39 in vivo and in vitro. Consistently, VapC inhibited translation in vivo and in vitro. Translation-reactions could be reactivated by the addition of VapB and extra charged tRNA(fMet). Similarly, ectopic production of tRNA(fMet) counteracted VapC in vivo. Thus, tRNA(fMet) is the only cellular target of VapC. Depletion of tRNA(fMet) by vapC induction was bacteriostatic and stimulated ectopic translation initiation at elongator codons. Moreover, addition of chloramphenicol to cells carrying vapBC induced VapC activity. Thus, by cleavage of tRNA(fMet), VapC simultaneously may regulate global cellular translation and reprogram translation initiation.

The stable maintenance of low-copy-number plasmids requires active partitioning, with the most common mechanism in prokaryotes involving the ATPase ParA. ParA proteins undergo intricate spatiotemporal relocations across the nucleoid, dynamics that function to position plasmids at equally spaced intervals. This spacing naturally guarantees equal partitioning of plasmids to each daughter cell. However, the fundamental mechanism linking ParA dynamics with regular plasmid positioning has proved difficult to dissect. In this issue of Molecular Microbiology, Vecchiarelli et al. report on a time-delay mechanism that allows a slow cycling between the nucleoid-bound and unbound forms of ParA. The authors also propose a mechanism for plasmid movement that does not rely on ParA polymerization.

In prokaryotes, DNA can be segregated by three different types of cytoskeletal filaments. The best-understood type of partitioning (par) locus encodes an actin homolog called ParM, which forms dynamically unstable filaments that push plasmids apart in a process reminiscent of mitosis. However, the most common type of par locus, which is present on many plasmids and most bacterial chromosomes, encodes a P loop ATPase (ParA) that distributes plasmids equidistant from one another on the bacterial nucleoid. A third type of par locus encodes a tubulin homolog (TubZ) that forms cytoskeletal filaments that move rapidly with treadmill dynamics., (Copyright 2010 Elsevier Inc. All rights reserved.)

FtsZ, the essential regulator of bacterial cell division, is a dynamic cytoskeletal protein that forms helices that condense into the Z-ring prior to division. Two small coiled-coil proteins, ZapA and ZapB, are both recruited early to the Z-ring. We show here that ZapB is recruited to the Z-ring by ZapA. A direct interaction between ZapA and ZapB is supported by bacterial two-hybrid and in vitro interaction assays. Using high-resolution 3-D reconstruction microscopy, we find that, surprisingly, ZapB is located inside the Z-ring in virtually all cells investigated. We propose a molecular model in which ZapA increases lateral interactions between FtsZ proto-filaments and ZapB mediates further stabilization of this interaction by cross-linking ZapA molecules bound to adjacent FtsZ proto-filaments. Gene deletion and complementation assays show that ZapB can mitigate cell division and Z-ring assembly defects even in the absence of ZapA, raising the possibility that ZapB stimulates Z-ring assembly by two different mechanisms.

Prokaryotic toxin - antitoxin (TA) loci encode mRNA interferases that inhibit translation, either by cleaving mRNA codons at the ribosomal A site or by cleaving any RNA site-specifically. So far, seven mRNA interferases of Escherichia coli have been identified, four of which cleave mRNA by a translation-dependent mechanism. Here, we experimentally confirmed the presence of three novel TA loci in E. coli. We found that the yafNO, higBA (ygjNM) and ygiUT loci encode mRNA interferases related to RelE. YafO and HigB cleaved translated mRNA only, while YgiU cleaved RNA site-specifically at GC[A/U], independently of translation. Thus, YgiU is the first RelE-related mRNA interferase that cleaves mRNA independently of translation, in vivo. All three loci were induced by amino acid starvation, and inhibition of translation although to different degrees. Carbon starvation induced only two of the loci. The yafNO locus was induced by DNA damage, but the transcription originated from the dinB promoter. Thus, our results showed that the different TA loci responded differentially to environmental stresses. Induction of the three loci depended on Lon protease that may sense the environmental stresses and activate TA loci by cleavage of the antitoxins. Transcription of the three TA operons was autoregulated by the antitoxins.

Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 A) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 A) and after (3.6 A) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2'-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.

RelB, the ribbon-helix-helix (RHH) repressor encoded by the relBE toxin-antitoxin locus of Escherichia coli, interacts with RelE and thereby counteracts the mRNA cleavage activity of RelE. In addition, RelB dimers repress the strong relBE promoter and this repression by RelB is enhanced by RelE; that is, RelE functions as a transcriptional co-repressor. RelB is a Lon protease substrate, and Lon is required both for activation of relBE transcription and for activation of the mRNA cleavage activity of RelE. Here we characterize the molecular interactions important for transcriptional control of the relBE model operon. Using an in vivo screen for relB mutants, we identified multiple nucleotide changes that map to important amino acid positions within the DNA-binding domain formed by the N-terminal RHH motif of RelB. Analysis of DNA binding of a subset of these mutant RHH proteins by gel-shift assays, transcriptional fusion assays and a structure model of RelB-DNA revealed amino acid residues making crucial DNA-backbone contacts within the operator (relO) DNA. Mutational and footprinting analyses of relO showed that RelB dimers bind on the same face of the DNA helix and that the RHH motif recognizes four 6-bp repeats within the bipartite binding site. The spacing between each half-site was found to be essential for cooperative interactions between adjacently bound RelB dimers stabilized by the co-repressor RelE. Kinetic and stoichiometric measurements of the interaction between RelB and RelE confirmed that the proteins form a high-affinity complex with a 2:1 stoichiometry. Lon degraded RelB in vitro and degradation was inhibited by RelE, consistent with the proposal that RelE protects RelB from proteolysis by Lon in vivo.

Bacterial plasmids encode partitioning (par) loci that confer stable plasmid inheritance. We showed previously that, in the presence of ParB and parC encoded by the par2 locus of plasmid pB171, ParA formed cytoskeletal-like structures that dynamically relocated over the nucleoid. Simultaneously, the par2 locus distributed plasmids regularly over the nucleoid. We show here that the dynamic ParA patterns are not simple oscillations. Rather, ParA nucleates and polymerizes in between plasmids. When a ParA assembly reaches a plasmid, the assembly reaction reverses into disassembly. Strikingly, plasmids consistently migrate behind disassembling ParA cytoskeletal structures, suggesting that ParA filaments pull plasmids by depolymerization. The perpetual cycles of ParA assembly and disassembly result in continuous relocation of plasmids, which, on time averaging, results in equidistribution of the plasmids. Mathematical modeling of ParA and plasmid dynamics support these interpretations. Mutational analysis supports a molecular mechanism in which the ParB/parC complex controls ParA filament depolymerization.

Toxin-antitoxin loci have been identified in almost all free-living prokaryotes, often in high copy numbers. The biological function and molecular targets of the abundant vapBC loci are not yet known. Here we analyse the vapBC loci of Salmonella LT2 and Shigella plasmid pMYSH6000. Both loci encode putative PIN (PilT N-terminal) domain toxins, and antitoxins that may regulate vapBC transcription. We show that vapBC(LT2) and vapBC(pMYSH) are bona fide TA loci: (i) both VapCs inhibited cell growth very efficiently and were counteracted by the cognate VapBs; (ii) both VapCs inhibited translation; (iii) transcription of the vapBC loci was induced by amino acid starvation and chloramphenicol, consistent with the proposal that VapB is an unstable inhibitor of vapBC transcription; (iv) ectopic expression of both VapCs induced a bacteriostatic condition that could be reversed by the cognate antitoxins. Unexpectedly, induction of vapC in Escherichia coli resulted in mRNA cleavage at stop-codons. Surprisingly, these cleavages depended on the yefM yoeB locus, indicating cross-activation between different toxins, that is, VapC activated YoeB mRNA interferase. Activation of YoeB depended on Lon, indicating that Lon degrades YefM antitoxin. Based on these results we present a model that explains activation of YoeB.

Background: The in vivo glucocorticoid response in childhood acute lymphoblastic leukaemia (ALL) correlates with the response to multi-agent chemotherapy. However, it is still unclear, whether the expression levels of glucocorticoid receptor (GR) splice variants facilitate the escape from glucocorticoid-induced apoptosis and hence contribute to glucocorticoid resistance., Procedure: In the present study, the initial in vivo expression of the common GR (cGR) and its splice variants GR-alpha, GR-gamma and GR-P was determined using a quantitative RT-PCR approach. Two cohorts of glucocorticoid sensitive (prednisone good responder, PGR) and resistant (prednisone poor responder, PPR) patients were compared. The kinetics of GR splice variant expression was measured during 36 hr following the first use of glucocorticoids in seven patients., Results: The GR splice variant GR-gamma showed the most pronounced differential regulation comparing PPR and PGR patients in both cohorts. GR-alpha and GR-gamma were upregulated faster and to a higher level in PGR as compared to PPR in the in vivo stimulation cohort. Here as well, the most pronounced effect was observed for GR-gamma., Conclusions: Differential regulation of the cGR and its splice variants under glucocorticoid treatment rather than the expression level at diagnosis is associated with glucocorticoid response., (Copyright 2008 Wiley-Liss, Inc.)

Toxin-antitoxin (TA) loci are common in free-living bacteria and archaea. TA loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin. The antitoxin can be either a protein or an antisense RNA. So far, six different TA gene families, in which the antitoxins are proteins, have been identified. Recently, Makarova et al. (K. S. Makarova, N. V. Grishin, and E. V. Koonin, Bioinformatics 22:2581-2584, 2006) suggested that the hicAB loci constitute a novel TA gene family. Using the hicAB locus of Escherichia coli K-12 as a model system, we present evidence that supports this inference: expression of the small HicA protein (58 amino acids [aa]) induced cleavage in three model mRNAs and tmRNA. Concomitantly, the global rate of translation was severely reduced. Using tmRNA as a substrate, we show that HicA-induced cleavage does not require the target RNA to be translated. Expression of HicB (145 aa) prevented HicA-mediated inhibition of cell growth. These results suggest that HicB neutralizes HicA and therefore functions as an antitoxin. As with other antitoxins (RelB and MazF), HicB could resuscitate cells inhibited by HicA, indicating that ectopic production of HicA induces a bacteriostatic rather than a bactericidal condition. Nutrient starvation induced strong hicAB transcription that depended on Lon protease. Mining of 218 prokaryotic genomes revealed that hicAB loci are abundant in bacteria and archaea.

Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation. This Phd domain is intrinsically disordered in solution and folds into an alpha-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin-antitoxin locus for its action in vivo.

Prokaryotic toxin-antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro but does not cleave pure RNA in vitro. RelE exhibits an incomplete RNase fold that may explain why RelE requires its substrate mRNA to presented by the ribosome. In contrast, RelE homologue YoeB has a complete RNase fold and cleaves RNA independently of ribosomes in vitro. Here, we show that YoeB cleavage of mRNA is strictly dependent on translation of the mRNA in vivo. Non-translated model mRNAs were not cleaved whereas the corresponding wild-type mRNAs were cleaved efficiently. Model mRNAs carrying frameshift mutations exhibited a YoeB-mediated cleavage pattern consistent with the reading frameshift thus giving strong evidence that YoeB cleavage specificity was determined by the translational reading frame. In contrast, site-specific mRNA cleavage by MazF occurred independently of translation. In one case, translation seriously influenced MazF cleavage efficiency, thus solving a previous apparent paradox. We propose that translation enhances MazF-mediated cleavage of mRNA by destabilization of the mRNA secondary structure.

Prokaryotic toxin-antitoxin (TA) loci consist of two genes in an operon that encodes a metabolically stable toxin and an unstable antitoxin. The antitoxin neutralizes its cognate toxin by forming a tight complex with it. In all cases known, the antitoxin autoregulates TA operon transcription by binding to one or more operators in the promoter region while the toxin functions as a co-repressor of transcription. Interestingly, the toxin can also stimulate TA operon transcription. Here we analyse mechanistic aspects of how RelE of Escherichia coli can function both as a co-repressor and as a derepressor of relBE transcription. When RelB was in excess to RelE, two trimeric RelB(2)*RelE complexes bound cooperatively to two adjacent operator sites in the relBE promoter region and repressed transcription. In contrast, RelE in excess stimulated relBE transcription and released the RelB(2)*RelE complex from operator DNA. A mutational analysis of the operator sites showed that RelE in excess counteracted cooperative binding of the RelB(2)*RelE complexes to the operator sites. Thus, RelE controls relBE transcription by conditional cooperativity.

Formation of the Z ring is the first known event in bacterial cell division. However, it is not yet known how the assembly and contraction of the Z ring are regulated. Here, we identify a novel cell division factor ZapB in Escherichia coli that simultaneously stimulates Z ring assembly and cell division. Deletion of zapB resulted in delayed cell division and the formation of ectopic Z rings and spirals, whereas overexpression of ZapB resulted in nucleoid condensation and aberrant cell divisions. Localization of ZapB to the divisome depended on FtsZ but not FtsA, ZipA or FtsI, and ZapB interacted with FtsZ in a bacterial two-hybrid analysis. The simultaneous inactivation of FtsA and ZipA prevented Z ring assembly and ZapB localization. Time lapse microscopy showed that ZapB-GFP is present at mid-cell in a pattern very similar to that of FtsZ. Cells carrying a zapB deletion and the ftsZ84(ts) allele exhibited a synthetic sick phenotype and aberrant cell divisions. The crystal structure showed that ZapB exists as a dimer that is 100% coiled-coil. In vitro, ZapB self-assembled into long filaments and bundles. These results raise the possibility that ZapB stimulates Z ring formation directly via its capacity to self-assemble into larger structures.

Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs.

In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insight into the workings of the par system from Escherichia coli plasmid R1. Despite its relative simplicity, the plasmid partition spindle shares characteristics with the mitotic machinery of eukaryotic cells.

The RelE and RelB proteins constitute the RNA interferase (toxin) and its cognate inhibitor (antitoxin) components of the Escherichia coli relBE toxin-antitoxin system. Despite the well-described functionality and physiological activity of this system in E. coli, no structural study was performed on the folding and stability of the protein pair in solution. Here we structurally and thermodynamically characterize the RelBE system components from E. coli in solution, both separately and in their complexed state. The RelB antitoxin, an alpha-helical protein according to circular dichroism and infrared spectroscopy, forms oligomers in solution, exhibits high thermostability with a TM of 58.5 degrees C, has a considerable heat resistance, and has high unfolding reversibility. In contrast, the RelE toxin includes a large portion of antiparallel beta-sheets, displays lower thermostability with a TM of 52.5 degrees C, and exhibits exceptional sensitivity to heat. Complex formation, accompanied by a structural transition, leads to a 12 degrees C increase in the TM and substantial heat resistance. Moreover, in vivo interaction and protein footprint experiments indicate that the C-terminal part of RelB is responsible for RelB-RelE interaction, being protease sensitive in its free state, while it becomes protected from proteolysis when complexed with RelE. Overall, our findings support the notion that RelB lacks a well-organized hydrophobic core in solution whereas RelE is a well-folded protein. Furthermore, our results support that RelB protein from E. coli is similar to ParD and CcdA antitoxins in both fold and thermodynamic properties. The differential folding state of the proteins is discussed in the context of their physiological activities.

Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA-binding protein ParR and its cognate centromere site parC on the DNA. The partition complex is recognized by a second partition protein, the actin-like ATPase ParM, which forms filaments required for the active bidirectional movement of DNA replicates. Here, we present the 2.8 A crystal structure of ParR from E. coli plasmid pB171. ParR forms a tight dimer resembling a large family of dimeric ribbon-helix-helix (RHH)2 site-specific DNA-binding proteins. Crystallographic and electron microscopic data further indicate that ParR dimers assemble into a helix structure with DNA-binding sites facing outward. Genetic and biochemical experiments support a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold. This structure holds implications for how ParM polymerization drives active DNA transport during plasmid partition.

The par2 locus of Escherichia coli plasmid pB171 encodes two trans-acting proteins, ParA and ParB, and two cis-acting sites, parC1 and parC2, to which ParB binds cooperatively. ParA is related to MinD and oscillates in helical structures and thereby positions ParB/parC-carrying plasmids regularly over the nucleoid. ParB ribbon-helix-helix dimers bind cooperatively to direct repeats in parC1 and parC2. Using four different assays we obtain solid evidence that ParB can pair parC1- and parC2-encoding DNA fragments in vitro. Convincingly, electron microscopy revealed that ParB mediates binary pairing of parC fragments. In addition to binary complexes, ParB mediated the formation of higher order complexes consisting of several DNA fragments joined by ParB at centromere site parC. N-terminal truncated versions of ParB still possessing specific DNA binding activity were incompetent in pairing, hence identifying the N terminus of ParB as a requirement for ParB-mediated centromere pairing. These observations suggest that centromere pairing is an important intermediate step in plasmid partitioning mediated by the common type I loci.

Recent genomic analyses revealed a surprisingly large number of toxin-antitoxin loci in free-living prokaryotes. The antitoxins are proteins or antisense RNAs that counteract the toxins. Two antisense RNA-regulated toxin-antitoxin gene families, hok/sok and ldr, are unrelated sequence-wise but have strikingly similar properties at the level of gene and RNA organization. Recently, two SOS-induced toxins were found to be regulated by RNA antitoxins. One such toxin, SymE, exhibits similarity with MazE antitoxin and, surprisingly, inhibits translation. Thus, it is possible that an ancestral antitoxin gene evolved into the present toxin gene (symE) whose translation is repressed by an RNA antitoxin (SymR).

The double par locus of Escherichia coli virulence factor pB171 consists of two adjacent and oppositely oriented par loci of different types, called par1 and par2. par1 encodes an actin ATPase (ParM), and par2 encodes an oscillating, MinD-like ATPase (ParA). The par loci share a central cis-acting region of approximately 200 bp, called parC1, located between the two par loci. An additional cis-acting region, parC2, is located downstream of the parAB operon of par2. Here we show that ParR of par1 and ParB of par2 bind cooperatively to unrelated sets of direct repeats in parC1 to form the cognate partition and promoter repression complexes. Surprisingly, ParB repressed transcription of the noncognate par operon, indicating cross-talk and possibly epistasis between the two systems. The par promoters, P1 and P2, affected each other negatively. The DNA binding activities of ParR and ParB correlated well with the observed transcriptional regulation of the par operons in vivo and in vitro. Integration host factor (IHF) was identified as a novel factor involved in par2-mediated plasmid partitioning.

Vibrio cholerae codes for 13 toxin-antitoxin (TA) loci all located within the superintegron on chromosome II. We show here that the two higBA TA loci of V. cholerae encode functional toxins, HigB-1 and HigB-2, whose ectopic expression inhibits cell growth of Escherichia coli, and functional antitoxins, HigA-1 and HigA-2, which counteract the toxicity of the cognate toxins. Three hours of ectopic expression of the HigB toxins resulted in bacteriostasis without any detectable loss of cell viability. The HigB toxins inhibited translation by cleavage of mRNA. Efficient mRNA cleavage occurred preferentially within the translated part of a model mRNA and only when the mRNA was translatable. Promoter analysis in V. cholerae and E. coli showed that the two higBA loci are both transcribed into bi-cistronic mRNAs and that the higBA-2 mRNA is leaderless. Transcription of the two higBA loci was strongly induced by amino acid (aa) starvation in V. cholerae and E. coli, indicating that the regulatory mechanisms of transcriptional induction are conserved across the two species. Both higBA loci stabilized a test-plasmid very efficiently in E. coli, raising the possibility that the loci contribute to maintain genetic stability of the V. cholerae superintegron. Based on these results we discuss the possible biological functions of the TA loci of V. cholerae.

Centromere-like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP-dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions without the involvement of partition-specific host cell receptors and is thus consistent with the striking observation that partition loci can function in heterologous host organisms.

Proteic toxin-antitoxin (TA) loci were first identified in bacterial plasmids, and they were regarded as involved in stable plasmid maintenance by a so-called 'addiction' mechanism. Later, chromosomally encoded TA loci were identified and their function ascribed to survival mechanisms when bacteria were subjected to stress. In the search for chromosomally encoded TA loci in Gram-positive bacteria, we identified various in the pathogen Streptococcus pneumoniae. Two of these cassettes, sharing homology with the Escherichia coli relBE locus were cloned and tested for their activity. The relBE2Spn locus resulted to be a bona fide TA locus. The toxin exhibited high toxicity towards E. coli and S. pneumoniae, although in the latter, the chromosomal copy of the antitoxin relB2Spn gene had to be inactivated to detect full toxicity. Cell growth arrest caused by expression of the relE2Spn toxin gene could be reverted by expression of the cognate antitoxin, relB2Spn, although prolonged exposition to the toxin led to cell death. The pneumococcal relBE2Spn locus is the first instance of a chromosomally encoded TA system from Gram-positive bacteria characterized in its own host. We have developed a bioluminescence resonance energy transfer (BRET) assay to detect the interactions between the RelB2Spn antitoxin and the RelE2Spn toxin in vivo. This technique has shown to be amenable to a high-throughput screening (HTS), opening new avenues in the search of molecules with potential antibacterial activity able to inhibit TA interactions.

Short regulatory RNAs are widespread in bacteria, and many function through antisense recognition of mRNA. Among the best studied antisense transcripts are RNA antitoxins that repress toxin mRNA translation. The hok/sok locus of plasmid R1 from Escherichia coli is an established model for RNA antitoxin action. Base-pairing between hok mRNA and Sok-antisense-RNA increases plasmid maintenance through post-segregational-killing of plasmid-free progeny cells. To test the model and the idea that sequestration of Sok-RNA activity could provide a novel antimicrobial strategy, we designed anti Sok peptide nucleic acid (PNA) oligomers that, according to the model, would act as competitive inhibitors of hok mRNA::Sok-RNA interactions. In hok/sok-carrying cells, anti Sok PNAs were more bactericidal than rifampicin. Also, anti Sok PNAs induced ghost cell morphology and an accumulation of mature hok mRNA, consistent with cell killing through synthesis of Hok protein. The results support the sense/antisense model for hok mRNA repression by Sok-RNA and demonstrate that antisense agents can be used to out-compete RNA::RNA interactions in bacteria. Finally, BLAST analyses of approximately 200 prokaryotic genomes revealed that many enteric bacteria have multiple hok/sok homologous and analogous RNA-regulated toxin-antitoxin loci. Therefore, it is possible to activate suicide in bacteria by targeting antitoxins.

The actin-like MreB cytoskeletal protein and RNA polymerase (RNAP) have both been suggested to provide the force for chromosome segregation. Here, we identify MreB and RNAP as in vivo interaction partners. The interaction was confirmed using in vitro purified components. We also present convincing evidence that MreB and RNAP are both required for chromosome segregation in Escherichia coli. MreB is required for origin and bulk DNA segregation, whereas RNAP is required for bulk DNA, terminus, and possibly also for origin segregation. Furthermore, flow cytometric analyses show that MreB depletion and inactivation of RNAP confer virtually identical and highly unusual chromosome segregation defects. Thus, our results raise the possibility that the MreB-RNAP interaction is functionally important for chromosome segregation.

Faithful chromosome segregation is vital to all organisms. Eukaryotic cells use the tubulin-based cytoskeleton to segregate their chromosomes during mitosis. A handful of papers have provided convincing evidence that, in bacteria, this task is accomplished by the actin homolog MreB. In particular, a recent study by Gitai et al. demonstrates that MreB specifically binds to and segregates the replication origin of the bacterial chromosome.

Summary Bacterial plasmids and chromosomes encode centromere-like partition loci that actively segregate DNA before cell division. The molecular mechanism behind DNA segregation in bacteria is largely unknown. Here we analyse the mechanism of partition-associated incompatibility for plasmid pB171, a phenotype associated with all known plasmid-encoded centromere loci. An R1 plasmid carrying par2 from plasmid pB171 was destabilized by the presence of an F plasmid carrying parC1, parC2 or the entire par2 locus of pB171. Strikingly, cytological double-labelling experiments revealed no evidence of long-lived pairing of plasmids. Instead, pure R1 and F foci were positioned along the length of the cell, and in a random order. Thus, our results raise the possibility that partition-mediated plasmid incompatibility is not caused by pairing of heterologous plasmids but instead by random positioning of pure plasmid clusters along the long axis of the cell. The strength of the incompatibility was correlated with the capability of the plasmids to compete for the mid-cell position.

Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.

Prokaryotic chromosomes code for toxin-antitoxin (TA) loci, often in multiple copies. In E.coli, experimental evidence indicates that TA loci are stress-response elements that help cells survive unfavorable growth conditions. The first gene in a TA operon codes for an antitoxin that combines with and neutralizes a regulatory 'toxin', encoded by the second gene. RelE and MazF toxins are regulators of translation that cleave mRNA and function, in interplay with tmRNA, in quality control of gene expression. Here, we present the results from an exhaustive search for TA loci in 126 completely sequenced prokaryotic genomes (16 archaea and 110 bacteria). We identified 671 TA loci belonging to the seven known TA gene families. Surprisingly, obligate intracellular organisms were devoid of TA loci, whereas free-living slowly growing prokaryotes had particularly many (38 in Mycobacterium tuberculosis and 43 in Nitrosomonas europaea). In many cases, TA loci were clustered and closely linked to mobile genetic elements. In the most extreme of these cases, all 13 TA loci of Vibrio cholerae were bona fide integron elements located in the V.cholerae mega-integron. These observations strongly suggest that TA loci are mobile cassettes that move frequently within and between chromosomes and also lend support to the hypothesis that TA loci function as stress-response elements beneficial to free-living prokaryotes.

MreB proteins of Escherichia coli, Bacillus subtilis and Caulobacter crescentus form actin-like cables lying beneath the cell surface. The cables are required to guide longitudinal cell wall synthesis and their absence leads to merodiploid spherical and inflated cells prone to cell lysis. In B. subtilis and C. crescentus, the mreB gene is essential. However, in E. coli, mreB was inferred not to be essential. Using a tight, conditional gene depletion system, we systematically investigated whether the E. coli mreBCD-encoded components were essential. We found that cells depleted of mreBCD became spherical, enlarged and finally lysed. Depletion of each mre gene separately conferred similar gross changes in cell morphology and viability. Thus, the three proteins encoded by mreBCD are all essential and function in the same morphogenetic pathway. Interestingly, the presence of a multicopy plasmid carrying the ftsQAZ genes suppressed the lethality of deletions in the mre operon. Using GFP and cell fractionation methods, we showed that the MreC and MreD proteins were associated with the cell membrane. Using a bacterial two-hybrid system, we found that MreC interacted with both MreB and MreD. In contrast, MreB and MreD did not interact in this assay. Thus, we conclude that the E. coli MreBCD form an essential membrane-bound complex. Curiously, MreB did not form cables in cell depleted for MreC, MreD or RodA, indicating a mutual interdependency between MreB filament morphology and cell shape. Based on these and other observations we propose a model in which the membrane-associated MreBCD complex directs longitudinal cell wall synthesis in a process essential to maintain cell morphology.

Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments that segregate plasmids paired at mid-cell to daughter cells. Like microtubules, ParM filaments exhibit dynamic instability (i.e., catastrophic decay) whose regulation is an important component of the DNA segregation process. The Walker box ParA ATPases are related to MinD and form highly dynamic, oscillating filaments that are required for the subcellular movement and positioning of plasmids. The role of the observed ATPase oscillation is not yet understood. However, we propose a simple model that couples plasmid segregation to ParA oscillation. The model is consistent with the observed movement and localization patterns of plasmid foci and does not require the involvement of plasmid-specific host-encoded factors.

The centromere sequence parC of Escherichia coli low-copy-number plasmid R1 consists of two sets of 11 bp iterated sequences. Here we analysed the intrinsic sequence-directed curvature of parC by its migration anomaly in polyacrylamide gels. The 159 bp long parC is strongly curved with anomaly values (k-factors) close to 2. The properties of the parC curvature agree with those of other curved DNA sequences. parC contains two regions of 5-fold repeated iterons separated by 39 bp. We modified 4 bp within this intermediate sequence so that we could analyse the two 5-fold repeated regions independently. The analysis shows that the two repeat regions are not independently curved parts of parC but that the overall curvature is a property of the whole fragment. Since the centromere sequence of an E.coli plasmid as well as eukaryotic centromere sequences show DNA curvature, we speculate that curvature might be a general property of centromeres.

Escherichia coli encodes two rel loci, both of which contribute to the control of synthesis of macromolecules during amino acid starvation. The product of relA (ppGpp synthetase I) is responsible for the synthesis of guanosine tetraphosphate, ppGpp, the signal molecule that exerts stringent control of stable RNA synthesis. The second rel locus, relBE, was identified by mutations in relB that confer a so-called 'delayed-relaxed response' characterized by continued RNA synthesis after a lag period of approximately 10 min after the onset of amino acid starvation. We show here that the delayed-relaxed response is a consequence of hyperactivation of RelE. As in wild-type cells, [ppGpp] increased sharply in relB101 relE cells after the onset of starvation, but returned rapidly to the prestarvation level. RelE is a global inhibitor of translation that is neutralized by RelB by direct protein-protein interaction. Lon protease activates RelE during amino acid starvation by degradation of RelB. We found that mutations in relB that conferred the delayed-relaxed phenotype destabilized RelB. Such mutations confer severe RelE-dependent inhibition of translation during amino acid starvation, indicating hyperactivation of RelE. Hyperactivation of RelE during amino acid starvation was shown directly by measurement of RelE-mediated cleavage of tmRNA. The RelE-mediated shutdown of translation terminated amino acid consumption and explains the rapid restoration of the ppGpp level observed in relB mutant cells. Restoration of the prestarvation level of ppGpp, in turn, allows for the resumption of stable RNA synthesis seen during the delayed-relaxed response.

The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells with a single plasmid focus, the focus located preferentially at mid-cell. In cells with two foci, these located at quarter-cell positions. In the absence of ParB and parC1/parC2, ParA-GFP formed stationary helices extending from one end of the nucleoid to the other. In the presence of ParB and parC1/parC2, ParA-GFP oscillated in spiral-shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid-cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid and subsequently separate them into daughter cells.